MD Atilla Yilmaz
email: atilla.yilmaz@uk-erlangen.de

MD Tina Schaller

PhD Iwona Cicha

Regina Altendorf

MD Christian Stumpf

MD Lutz Klinghammer

MD Josef Ludwig

MD Werner G Daniel

MD Chris- Toph D Garlichs

Predictive value of the decrease in circulating dendritic cell precursors in stable coronary artery disease

Keywords: atherosclerosis, coronary artery disease, immune system, dendritic cells Circulating DC precursors in stable CAD

Dendritic cells are present in atherosclerotic lesions leading to vascular inflammation. The number of vascular dendritic cells increases during atherosclerosis. Recently, we showed that circulating dendritic cell precursors (DCP) are reduced in acute coronary syndromes through vascular recruitment. In our present study, we investigated whether DCP might be reduced in stable coronary artery disease (CAD) too. The levels of circulating myeloid (mDCP), plasmacytoid (pDCP), and total DCP (tDCP) were flow cytometrically investigated in 290 patients with suspected stable CAD. The coronary angiogram was used to evaluate for each patient a CAD Score: 1. excluded CAD (n=57), 2. early CAD (n=63), 3. moderate CAD (n=85), and 4. advanced CAD (n=85). Compared to controls, patients with advanced stable CAD had lower HDL cholesterol (p = 0.03) and higher creatinine (p = 0.003). In advanced CAD a significant decrease of circulating mDCP, pDCP, and tDCP was shown (each P < 0.001). A significant inverse correlation was observed between the CAD Score and mDCP, pDCP, or tDCP (each P < 0.001). Patients who required a percutaneous coronary intervention or coronary artery bypass grafting had less circulating mDCP, pDCP, and tDCP than controls (each p < 0.001). Multiple stepwise logistic regression analysis proved mDCP, pDCP, and tDCP as independent predictors of a CAD. In conclusion, we show that patients with stable CAD have significantly lower levels of circulating DCP than healthy individuals. Their decrease seems to be an independent predictor of the presence and consecutive therapeutical procedure in stable CAD.

I N T R O D U C T I O N

Atherosclerosis is a chronic inflammatory disease characterized by the invasion of immune cells such as T cells, monocytes, dendritic cells (DC), and mast cells into the vascular wall [START_REF] Hansson | Inflammation, atherosclerosis, and coronary artery disease[END_REF]. A decade ago, DC were firstly described in atherosclerotic lesions as well as healthy vascular intima [START_REF] Bobryshev | S-100 positive cells in human arterial intima and in atherosclerotic lesions[END_REF][START_REF] Millonig | Network of vascular-associated dendritic cells in intima of healthy young individuals[END_REF]. DC are professional antigen-presenting cells with the unique ability to stimulate naïve as well as memory T cells [START_REF] Banchereau | Immunobiology of dendritic cells[END_REF]. Therefore, DC play a critical role in many inflammatory conditions, particularly in autoimmune diseases [START_REF] Cravens | Dendritic cells, chemokine receptors and autoimmune inflammatory diseases[END_REF]. DC patrol the blood and the peripheral tissues to detect foreign, potential harmfull antigens, able to initiate an immune response against them [START_REF] Liu | Dendritic cell subsets and lineages, and their functions in innate and adaptive immunity[END_REF]. Two lineages of DC can be differentiated: Myeloid DC (CD1c + , CD11c + ) which respond to bacteria and fungi, releasing IL-12, as well as plasmacytoid DC (CD303 + ) which release interferon-upon viral infection [START_REF] Liu | Dendritic cell subsets and lineages, and their functions in innate and adaptive immunity[END_REF]. Both lineages can be detected in blood as DC precursors (DCP) which possess a high expression of HLA-DR, but lack the expression of costimulatory molecules, so that they are not able to activate T cells. After invasion of (inflamed) tissue they change their morphological appearance, and are enabled to take up potential antigens (immature DC), subsequently acquiring the ability to stimulate in an antigen-specific manner T cells through upregulation of costimulatory molecules (mature DC).

In recent times, it was suggested that myeloid DC are critically involved in the progression and destabilization of atherosclerotic plaques [START_REF] Yilmaz | Emergence of dendritic cells in ruptureprone regions of vulnerable carotid plaques[END_REF][START_REF] Erbel | Functional profile of activated dendritic cells in unstable atherosclerotic plaque[END_REF][START_REF] Kawahara | The expression of vascular dendritic cells in human atherosclerotic carotid plaques[END_REF][START_REF] Bobryshev | Mapping of vascular dendritic cells in atherosclerotic arteries suggests their involvement in local immune-inflammatory reactions[END_REF]. In the vessel wall, myeloid DC are activated by different atherogenic factors, such as oxidized low density lipoprotein (LDL) cholesterol [START_REF] Alderman | Effects of oxidised low density lipoprotein on dendritic cells: a possible immunoregulatory component of the atherogenic micro-environment?[END_REF], nicotine [START_REF] Aicher | Nicotine strongly activates dendritic cell-mediated adaptive immunity: potential role for progression of atherosclerotic lesions[END_REF], or advanced glycation end products [START_REF] Ge | Advanced glycosylation end products might promote atherosclerosis through inducing the immune maturation of dendritic cells[END_REF], enabling them to stimulate T cells, and probably leading to vascular inflammation. Proving this concept, Han et al. recently demonstrated in a vascular model that myeloid DC are the most potent T cell activators in atherosclerosis and crucial for the immune reactions which occur in the vessel wall microenvironment [START_REF] Han | Vessel wall-embedded dendritic cells induce T-cell autoreactivity and initiate vascular inflammation[END_REF]. A c c e p t e d M a n u s c r i p t

Whereas scientific investigations had focussed on the role of myeloid DC in atherosclerosis so far, Niessner et al. recently described that plasmacytoid DC are present in atherosclerotic plaques too, and that they stimulate T cells against viral antigens there [START_REF] Niessner | Pathogen-sensing plasmacytoid dendritic cells stimulate cytotoxic T-cell function in the atherosclerotic plaque through interferonalpha[END_REF].

During the progression of atherosclerosis, the number of mature myeloid and plasmacytoid DC significantly increases in atherosclerotic lesions, and two independent mechanisms are responsible for that. First, in atherogenic areas of the vascular wall an increased recruitment of circulating DCP was observed with accumulation particularly at the upstream shoulder of atherosclerotic plaques [START_REF] Yilmaz | Decrease in circulating myeloid dendritic cell precursors in coronary artery disease[END_REF][START_REF] Yilmaz | Accumulation of immune cells and high expression of chemokines/chemokine receptors in the upstream shoulder of atherosclerotic carotid plaques[END_REF]. Certain atherogenic factors such as hypoxia, oxidized LDL cholesterol, or tumor necrosis factor (TNF)-seem to enhance the transmigration of DCP over the endothelium into the intima [START_REF] Weis | Endothelial determinants of dendritic cell adhesion and migration: new implications for vascular diseases. Arterioscler[END_REF]. Second, recent studies showed that the emigration of DC from atherosclerotic lesions into the regional lymph nodes is reduced by certain pro-atherogenic conditions such as the presence of oxidized LDL cholesterol or platelet-activating factor [START_REF] Angeli | Dyslipidemia associated with atherosclerotic disease systemically alters dendritic cell mobilization[END_REF][START_REF] Llodra | Emigration of monocyte-derived cells from atherosclerotic lesions characterizes regressive, but not progressive, plaques[END_REF]. Activated platelets were shown to induce both vascular recruitment and maturation of circulating DCP [START_REF] Langer | Platelets recruit human dendritic cells via Mac-1/JAM-C interaction and modulate dendritic cell function in vitro[END_REF].

In two recent studies it was firstly described that the level of circulating DCP is significantly reduced in blood of patients with acute coronary syndrome (ACS), indicating the possibility of their increased recruitment into the vascular wall [START_REF] Yilmaz | Decrease in circulating myeloid dendritic cell precursors in coronary artery disease[END_REF][START_REF] Van Vre | Decreased number of circulating plasmacytoid dendritic cells in patients with atherosclerotic coronary artery disease[END_REF]. Patients with stable coronary artery disease (CAD) seemed to have a reduction of circulating DCP too [START_REF] Yilmaz | Decrease in circulating myeloid dendritic cell precursors in coronary artery disease[END_REF], implicating that their decrease might be not only a transient effect in ACS but could also represent a permanent state in stable CAD. Beyond alterations in the frequency of circulating DCP it was recently shown that in patients with CAD, particularly those with ACS, cultivated DC from blood monocytes had a significantly higher expression of costimulatory molecules, suggesting a higher ability of them to stimulate T cells [START_REF] Dopheide | Monocyte-derived dendritic cells of patients with coronary artery disease show an increased expression of costimulatory molecules CD40, CD80 and CD86 in vitro[END_REF]. It was suggested that differentiation of monocytes into myeloid DC might be an alternative way for their increase in the atherosclerotic altered vascular wall [START_REF] Dopheide | Monocyte-derived dendritic cells of patients with coronary artery disease show an increased expression of costimulatory molecules CD40, CD80 and CD86 in vitro[END_REF].

M E T H O D S Study population

Between December 2005 and July 2007, 290 patients with suspected CAD who underwent a coronary angiography at our clinic were enrolled in this study. Exclusion criteria were acute coronary syndromes (unstable angina pectoris, new or dynamic ST segment changes, and/or troponin-I > 0.5 ng/mL) and non-cardiac diseases that could interfere with our analysis: acute or chronic infections, malignancies, autoimmune diseases, hyperthyroidism, and medication with immunosuppressive agents. Routine blood analyses were performed in our hospital laboratory according to clinic standard. Standardized blood sampling for flow cytometrical analysis was carried out at admission before coronary angiography and in some of the patients after 24h. Immediately after blood withdrawal, samples were flow cytometrically analyzed. The study was carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association. Informed consent was obtained from all patients and the study was approved by the institutional ethics committee.

Evaluation of the CAD Score and subdivision into the study groups

For each patient, a CAD Score was calculated. For this purpose, the coronary angiogram was analyzed by two experienced investigators, and for each of 15 coronary segments the maximum grade of stenosis was assessed as previously described: left main coronary artery (S5), the proximal (S6), middle (S7), and distal (S8) left anterior descending (LAD), the first (S9) and second (S10) diagonal branch of the LAD, the proximal (S11), middle (S13) A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. and distal (S15) circumflex artery (CX), the first (S12) and second (S14) obtuse marginal branch of the CX, and the proximal (S1), medial (S2), and distal (S3 & S4) right coronary artery (RCA). According to the maximum grade of stenosis, each segment received a point value: 0% (0 point), 1-25% (1 point), 26-50% (2 points), 51-75% (3 points), and > 75% (4 points) [START_REF] Austen | A reporting system on patients evaluated for coronary artery disease. Report of the Ad Hoc Committee for Grading of Coronary Artery Disease[END_REF]. The CAD Score was calculated for each patient by counting up the point values in each coronary segment. According to the CAD Score, patients were subdivided into the following groups: Score 0 (excluded CAD, n=57), Score 1-5 (early CAD, n=63), Score 6-10 (moderate CAD, n=85), and Score > 10 (advanced CAD, n=85). Additionally, patients were subdivided according to the therapeutical recommendation into the following groups: 1. no therapy required (n=48), 2. medication (n=123), 3. percutaneous coronary intervention (PCI, n=80), or 4. coronary artery bypass grafting (CABG, n=44).

Serum analysis of high-sensitivity CRP.

The serum concentration of high-sensitivity (hs)CRP was measured using an immunonephelometric assay on a BN II analyzer (Dade Behring, Marburg, Germany).

Identification of DC precursors by fluorescence activated cell sorting (FACS)

The technique for the analysis of circulating mDCP and pDCP was developed by Ito et al. [START_REF] Ito | A CD1a+/CD11c+ subset of human blood dendritic cells is a direct precursor of Langerhans cells[END_REF] and Dzionek et al. [START_REF] Dzionek | BDCA-2, BDCA-3, and BDCA-4: three markers for distinct subsets of dendritic cells in human peripheral blood[END_REF] who showed that FACS sorting of BDCA-1 + and BDCA-2 + cells led to the isolation of cells who express mDC-and pDC-specific markers, respectively, and possess certain functions which are characteristic of DC. This technique is provided in the Blood Dendritic Cell Enumeration kit TM (BDCA kit, Miltenyi Biotec, Bergisch Gladbach, Germany) which was used in our study.

Fresh blood samples were collected on EDTA. Three color-staining and FACS analysis were performed as previously described [START_REF] Yilmaz | Decrease in circulating myeloid dendritic cell precursors in coronary artery disease[END_REF]. Briefly, 300 µL of blood was mixed with 20 µL of the anti-BDCA-Cocktail for DCP analysis and another 300 µL of blood was A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. mixed with 20 µL of the control cocktail for the isotype control. Subsequently, 10 µL of dead cell discriminator was added, and the samples were incubated for 10 min. under a 60W light bulb. Erythrocytes were removed using the red blood cell lysis solution, following the fixation of the samples with the fix solution.

FACS analysis was performed using the FACSCalibur flow cytometer with Cell-Quest software (Becton Dickinson). Since circulating DCP comprise only 0.1 to 1% of white blood cells (WBC), a special gating strategy was used to analyze mDCP, pDCP, and tDCP accurately (Fig. 1). For each sample, 2x10 5 WBC were registered in region (R)1 defined by forward scatter (FSC) and side scatter (SSC). In R2, granulocytes were excluded according to their high SSC, and monocytes, lymphocytes, and dead cells were excluded according to their CD14-, CD19-, and propidium iodide-staining, respectively. In R3 and R4, circulating mDCP and pDCP were detected according to their specific staining for BDCA-1 and BDCA-2, respectively, and tDCP were the sum of cells in R3 and R4. The relative cell numbers of circulating mDCP, pDCP, and tDCP were assessed as a percentage of WBC (% WBC). The absolute cell numbers (cells/µL) were calculated using their relative cell numbers multiplied by the white blood cell count.

Statistical analysis

Statistical analyses were performed using the SigmaStat 2.03 software of the SPSS incorporation (Chicago, U.S.A.). Data were expressed as median or as proportions (%). P < 0.05 was considered statistically significant. The non-parametric Mann-Whitney rank sum test was used to compare numerical data (e.g. mDCP, pDCP, or tDCP values) between the study groups as well as for the subgroup analysis. Categorical clinical data (e.g. sex, atherogenic risk factors, medication) were compared between the study groups by Chisquare test. Correlation analyses, e.g. circulating DCP with the CAD Score, were performed using the non-parametric Spearman rank order test. The predictive value of different parameters for a CAD was analyzed using univariate and multivariate logistic regression analysis. For this purpose, CAD was encoded binary for each patient (all coronary stenosis < 50%: "0"; one or more coronary stenoses > 50%: "1"), and was analyzed as dependent variable compared with other factors (e.g. mDCP, pDCP, or tDCP values) as independent variables. Quantitative variables such as DCP values, creatinine, or age, were binary encoded for the logistic regression analysis too using the median calculated for all patients (individual value < median: "0"; individual value > median: "1"). Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. Since mDCP, pDCP, and tDCP showed a high correlation with each other, for each of them a separate multiple stepwise logistic regression analysis was performed including all parameters of the univariate analysis.

R E S U L T S Baseline characteristics

Clinical characteristics of patients with coronary artery disease (CAD) are reported in Table 1. Compared to individuals without CAD, patients with CAD were significantly older (p < 0.001), more often treated with statins (p < 0.001) or aspirin (p = 0.03), and had lower high density lipoprotein (HDL) cholesterol (p = 0.03) and higher creatinine (p = 0.003).

Additionally, more often diabetes mellitus and hyperlipidemia as well as a trend for higher hsCRP and leukocytes were observed in patients with a CAD. Regarding the therapeutical procedure suggested in the coronary angiogram report, patients with mild CAD (Score 1-5) rarely required a PCI (16%), PCI was performed in 41% of patients with moderate CAD (Score 6-10), whereas only 8% of them were treated with CABG. In advanced CAD (Score > 10) nearly equal numbers of patients underwent a PCI (39%) or CABG (44%).

Decrease in circulating DCP according to the stage of CAD

Comparing patients with mild (CAD Score 1-5), moderate (CAD Score 6-10), or advanced CAD (CAD Score > 10) with coronary healthy individuals (CAD Score 0), a stagedependent significant decrease in circulating mDCP, pDCP, and tDCP was observed. (Fig. 2A-F, for exact values see Table 1). Since relative as well as absolute numbers of circulating DCP were reduced, we were able to exclude the possibility that their decrease was caused by a dilution phenomenon due to an increase of another WBC population.

In a part of the study patients, a second DCP analysis was performed after 24h, showing that coronary angiography (n = 51) or PCI (n = 38) itself did not cause any alteration in the level of circulating DCP (data not shown).

Decrease in circulating DCP according to the required therapeutical procedure

In a second model, patients were subdivided into four different study groups according to the therapeutical procedure suggested in the coronary angiogram report: (1) Excluded CAD with no necessity of any therapy compared to CAD to be treated with (2) medication, (3) PCI, or (4) CABG. Compared with control individuals or CAD patients suggested to medication, CAD patients requiring a CABG or PCI had significantly reduced relative and absolute numbers of circulating mDCP, pDCP, and tDCP (Fig. 3A-F).

Effect of atherogenic risk factors or current medication on circulating DCP levels

In a subgroup analysis (table 2), we investigated whether different clinical factors or preexisting medical therapy might influence the levels of circulating DCP. Regarding age and gender of the patients, no significant changes were observed. In patients with hypertension or hyperlipidemia significantly lower levels of circulating pDCP and tDCP, and a trend for lower mDCP, were observed. Patients with diabetes mellitus had a trend for lower levels of mDCP, pDCP, and tDCP too. No significant changes were observed for smoking or family LDL cholesterol, HDL cholesterol, hsCRP, or creatinine were not associated with any changes in circulating DCP. As expected, higher blood leukocytes were associated with higher DCP levels.

Correlation of circulating DCP and further serum parameters with the CAD Score.

To analyze if circulating DCP and other serum parameters are directly associated with a CAD, correlation analyses were performed. These analyses showed a significant inverse correlation between the CAD Score and circulating mDCP% (Fig. 4A), pDCP% (Fig. 4B), and tDCP% (Fig. 4C). Further parameters which were significantly, but weaker correlated with the CAD Score were: HDL cholesterol (Fig. 4E) and creatinine (Fig. 4F). For hsCRP, only a trend for correlation with the CAD Score was observed (Fig. 4D). However, an inverse correlation was observed between hsCRP and circulating mDCP (r = -0.27; p < 0.001), pDCP (r = -0.19; p < 0.002), and tDCP (r = -0.25; p = 0.001). Comparing the different DC subtypes, circulating mDCP and pDCP showed a strong significant correlation with each other (r = 0.55; p < 0.001).

Circulating DCP as independent predictors of the presence of a CAD

As shown in Table 1 and Fig. 4, different factors seemed to be associated with the presence of a CAD. Thus, we investigated next which factors might have a predictive value for a CAD. Univariate analyses showed that significant predictors of a CAD were age > 66 years, diabetes mellitus, HDL cholesterol < 46.5 mg/dL, and hsCRP > 1.97 mg/dL (Fig. 5A). Additionally, the reduction of circulating mDCP < 0.21% or < 15.2/µL, pDCP < 0.10% or < 7.9/µL, and tDCP < 0.33% or < 24.8/µL were also predictive of a CAD, and actually seemed to be the strongest predictor (Fig. 5A). Male gender, hypertension, and

D I S C U S S I O N

Myeloid DC were implicated in the inflammatory processes related to atherosclerosis [START_REF] Yilmaz | Decrease in circulating myeloid dendritic cell precursors in coronary artery disease[END_REF][START_REF] Bobryshev | Dendritic cells in atherosclerosis: current status of the problem and clinical relevance[END_REF]. It was shown that they are present in atherosclerotic plaques, that their number increases in the vascular wall depending on the stage of atherosclerosis, and that they may promote plaque destabilization [START_REF] Yilmaz | Emergence of dendritic cells in ruptureprone regions of vulnerable carotid plaques[END_REF][START_REF] Erbel | Functional profile of activated dendritic cells in unstable atherosclerotic plaque[END_REF][START_REF] Kawahara | The expression of vascular dendritic cells in human atherosclerotic carotid plaques[END_REF]. Apart from that, recent studies independently showed that the level of circulating DCP is reduced in blood of patients with a CAD, particularly of those with ACS [START_REF] Yilmaz | Decrease in circulating myeloid dendritic cell precursors in coronary artery disease[END_REF][START_REF] Van Vre | Decreased number of circulating plasmacytoid dendritic cells in patients with atherosclerotic coronary artery disease[END_REF]. The aim of our present study was to investigate whether the level of circulating DCP is altered in stable coronary artery disease too. Compared to previous studies [START_REF] Yilmaz | Decrease in circulating myeloid dendritic cell precursors in coronary artery disease[END_REF][START_REF] Van Vre | Decreased number of circulating plasmacytoid dendritic cells in patients with atherosclerotic coronary artery disease[END_REF], circulating DCP were analyzed in a higher number of patients with angiographically exactly evaluated stable CAD, allowing us to investigate whether the decrease in circulating DCP is associated with the stage of CAD, or any other clinical factors, and if their decrease is an independent predictor of coronary artery stenoses.

In the present study, we show a stage-dependent decrease in circulating mDCP, pDCP, and tDCP in patients with a CAD compared to individuals with similar age, coronary risk factors, and angiographically excluded CAD. In contrast to our previous study [START_REF] Yilmaz | Decrease in circulating myeloid dendritic cell precursors in coronary artery disease[END_REF],

where we described a predominant decrease in circulating mDCP in unstable CAD and A c c e p t e d M a n u s c r i p t acute ST-elevation myocardial infarction (STEMI), we now found in patients with exclusively stable CAD a comparable decrease in both circulating mDCP and pDCP, as reported by van Vre et al. recently [START_REF] Van Vre | Decreased number of circulating plasmacytoid dendritic cells in patients with atherosclerotic coronary artery disease[END_REF]. The explanation for the discrepancy concerning the DCP results in stable CAD and STEMI seems to lie in the different migratory pattern of mDCP and pDCP. Whereas mDCP are recruited to the sites of inflammation in response to inflammatory chemokines, pDCP are attracted steadily by homeostatic chemokines into the lymphatic tissue [START_REF] Penna | Differential migration behavior and chemokine production by myeloid and plasmacytoid dendritic cells[END_REF]. Proving this concept, in a previous study in patients with dengue fever stable levels of circulating pDCP but a significant reduction of circulating mDCP due to enhanced emigration into inflammatory lesions was shown [START_REF] Pichyangkul | A blunted blood plasmacytoid dendritic cell response to an acute systemic viral infection is associated with increased disease severity[END_REF]. Indeed, we recently found in necrotic myocardium of patients with STEMI a dense infiltration with mDC but not pDC (unpublished data). In the study of Van Vre et al. [START_REF] Van Vre | Decreased number of circulating plasmacytoid dendritic cells in patients with atherosclerotic coronary artery disease[END_REF], patients with STEMI were excluded similar to our present investigation, explaining the comparable DCP decrease pattern observed in both these studies. In a recently published work, Van Vre et al. described that mDC are activated by hsCRP leading to DC maturation and increased DC-induced T cell stimulation, and that mDC are colocalized with hsCRP in atherosclerotic lesions [START_REF] Van Vre | Decreased number of circulating plasmacytoid dendritic cells in patients with atherosclerotic coronary artery disease[END_REF].

Corresponding to that, we observed in the present study a significant correlation between circulating DCP, particularly mDCP, and serum hsCRP, indicating that hsCRP might be involved in mDCP recruitment into atherosclerotic lesions in CAD.

Regarding the therapeutical procedure, we compared CAD patients who required CABG or PCI with those appointed to medication, or no need for any treatment. Patients who required CABG or PCI had significantly lower levels of circulating mDCP, pDCP, and tDCP compared to the other groups, indicating that their level might be predictive of the targeted therapy after coronary angiography. Skowasch et al. described a recruitment of mDCP into the treated lesion after PCI, suggesting that they are responsible for in-stent restenosis [START_REF] Skowasch | Presence of bone-marrow-and neuralcrest-derived cells in intimal hyperplasia at the time of clinical in-stent restenosis[END_REF]. However, in our study we were not able to detect any differences in the level of circulating DCP in PCI-treated patients before and 24 h after PCI. Compared to other previously published studies [START_REF] Yilmaz | Decrease in circulating myeloid dendritic cell precursors in coronary artery disease[END_REF][START_REF] Van Vre | Decreased number of circulating plasmacytoid dendritic cells in patients with atherosclerotic coronary artery disease[END_REF], the higher number of patients in our present study allowed us to perform a detailed subgroup analysis. We showed significantly lower levels of circulating pDCP and tDCP, and a trend for lower mDCP, in patients with hypertension or hyperlipidemia. Patients with diabetes mellitus had a trend for lower mDCP, pDCP, and tDCP too. An important aspect of the subgroup analysis is that patients with a family history of a CAD had no reduced levels of circulating DCP, indicating that lower levels of DCP are not a genetically determined factor for the development of a CAD. A trend for lower DCP levels were observed in patients treated with statins, aspirin, blockers. However, regarding table 1 a significantly higher number of CAD patients than controls were treated with aspirin or statins, so that the higher percentage of CAD patients in the corresponding treatment groups (table 2) is most likely to be responsible for the observed slightly lower levels of circulating DCP in those groups, and a drug-induced effect can be excluded.

Several factors were described to be predictive of the presence of significant coronary stenoses in patients with suspected CAD and of the cardiovascular risk in patients with known CAD. It was shown that lipoprotein(a) [START_REF] Mohan | Lipoprotein(a) is an independent risk factor for coronary artery disease in NIDDM patients in South India[END_REF][START_REF] Saely | Lipoprotein(a), type 2 diabetes and vascular risk in coronary patients[END_REF], lipoprotein-associated phospholipase A2 [START_REF] May | Lipoprotein-associated phospholipase A2 independently predicts the angiographic diagnosis of coronary artery disease and coronary death[END_REF], total and differential leukocyte counts [START_REF] Rasouli | Total and differential leukocytes counts, but not hsCRP, ESR, and five fractioned serum proteins have significant potency to predict stable coronary artery disease[END_REF], leptin [START_REF] Hoefle | Leptin, leptin soluble receptor and coronary atherosclerosis[END_REF], serum glycated albumin [START_REF] Pu | Increased serum glycated albumin level is associated with the presence and severity of coronary artery disease in type 2 diabetic patients[END_REF], and hsCRP [START_REF] Pu | Value of serum glycated albumin and highsensitivity C-reactive protein levels in the prediction of presence of coronary artery disease in patients with type 2 diabetes[END_REF] are significantly associated with and predictive of a stable CAD. In those studies it was shown in univariate analyses that certain clinical and serological factors such as age, male sex, diabetes mellitus, hypertension, HDL cholesterol, creatinine, and hsCRP were predictors of the presence of a stable CAD [START_REF] Mohan | Lipoprotein(a) is an independent risk factor for coronary artery disease in NIDDM patients in South India[END_REF][START_REF] Rasouli | Total and differential leukocytes counts, but not hsCRP, ESR, and five fractioned serum proteins have significant potency to predict stable coronary artery disease[END_REF][START_REF] Pu | Increased serum glycated albumin level is associated with the presence and severity of coronary artery disease in type 2 diabetic patients[END_REF][START_REF] Pu | Value of serum glycated albumin and highsensitivity C-reactive protein levels in the prediction of presence of coronary artery disease in patients with type 2 diabetes[END_REF].

However, most of those factors are associated with each other, so that even established atherogenic markers such as hsCRP, interleukin (IL)-6, and TNF-lost their independent predictive value for a stable CAD in multivariate analysis after adjusting for conventional CAD risk factors [START_REF] Sukhija | Inflammatory markers, angiographic severity of coronary artery disease, and patient outcome[END_REF]. In contrast, large-scale clinical studies showed that hsCRP, IL-6, A c c e p t e d M a n u s c r i p t and several other factors are well established predictors of future ischemic cardiovascular events in patients with higher risk for or a known CAD [START_REF] Ridker | Plasma concentration of interleukin-6 and the risk of future myocardial infarction among apparently healthy men[END_REF][START_REF] Albert | Prospective study of C-reactive protein, homocysteine, and plasma lipid levels as predictors of sudden cardiac death[END_REF].

In our study, we investigated whether the decrease in circulating DCP is predictive of the presence of a stable CAD. In the first step, we investigated which factors are associated with a stable CAD using correlation analysis. Compared to hsCRP, HDL, and creatinine, we showed a higher significant correlation of circulating mDCP, pDCP, and tDCP with the CAD score. Subsequently, we performed logistic regression analyses to investigate if the decrease in circulating DCP is a predictor of the presence of significant coronary artery stenoses. In univariate analysis, several factors such as higher age, diabetes mellitus, low HDL cholesterol, high hsCRP, and reduced relative and absolute levels of circulating mDCP, pDCP, and tDCP were predictors of a CAD. Male gender, hypertension, and elevated creatinine seem to have a predictive value too, but did not reach statistical significance. In multivariate analyses only the reduction of circulating mDCP, pDCP, or tDCP remained as independent predictors of a significant CAD.

The reason for the decrease in circulating DCP could not be clarified in our study.

Several explanations are possible such as (1) reduced production of DCP in the bone marrow, (2) increased apoptosis of circulating DCP, or (3) enhanced recruitment and higher turnover of circulating DCP in atherosclerotic lesions. There is no evidence for the first or the second explanation as other leukocyte subpopulations are not reduced in blood of CAD patients. There exist several studies supporting the third explanation. Firstly, it was shown that mDC and pDC accumulate in vascular lesions dependent on the stage of atherosclerosis [START_REF] Yilmaz | Emergence of dendritic cells in ruptureprone regions of vulnerable carotid plaques[END_REF][START_REF] Erbel | Functional profile of activated dendritic cells in unstable atherosclerotic plaque[END_REF][START_REF] Kawahara | The expression of vascular dendritic cells in human atherosclerotic carotid plaques[END_REF]. Secondly, it was suggested that mDCP recruitment from blood into the intima is induced by several atherogenic factors like oxidized LDL cholesterol, hypoxia, TNF-, or nicotine [START_REF] Aicher | Nicotine strongly activates dendritic cell-mediated adaptive immunity: potential role for progression of atherosclerotic lesions[END_REF][START_REF] Weis | Endothelial determinants of dendritic cell adhesion and migration: new implications for vascular diseases. Arterioscler[END_REF]. Thirdly, recirculation of mDC from the vascular wall into blood is suppressed by lysophosphatidic acid or platelet-activating factor in atherosclerosis [START_REF] Angeli | Dyslipidemia associated with atherosclerotic disease systemically alters dendritic cell mobilization[END_REF]. In conclusion, the results of our present study show a significant decrease in circulating mDCP, pDCP, and tDCP in patients with a CAD with stable clinical manifestation. The extent of their reduction is associated with the stage and with the recommended therapeutical procedure of CAD. Atherogenic risk factors such as hypertension or hyperlipidemia might affect the level of circulating DCP too. In multiple stepwise logistic regression analysis we prove that in contrast to other classical predictors only the decrease in circulating mDCP, pDCP, and tDCP remains as an independent predictor of a significant CAD. Thus, analysis of circulating DCP might be useful for the diagnosis of CAD. Future prospective studies will be necessary to investigate if the decrease in circulating DCP is predictive also of future ischemic cardiovascular events in patients with high risk for or known CAD.

A C K N O W L E D G E M E N T S

This work was supported by the Interdisciplinary Center for Clinical Research of the University Hospital of Erlangen. We thank Miss Katja Schubert and Mrs. Doris Flick for technical assistance. A c c e p t e d M a n u s c r i p t

D I S C L O S U R E S

F I G U R E L E G E N D S

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

Clinical

  Science Immediate Publication. Published on 23 Sep 2008 as manuscript CS20080392 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080392

Clinical

  Science Immediate Publication. Published on 23 Sep 2008 as manuscript CS20080392 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080392 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. The aim of our present study was to analyze in a larger-scale clinical investigation whether circulating myeloid or plasmacytoid DCP are significantly reduced in stable CAD, which factors are associated with their reduction, and to determine if the analysis of circulating mDCP or pDCP can predict the presence and stage of a CAD.

Clinical

  Science Immediate Publication. Published on 23 Sep 2008 as manuscript CS20080392 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080392

Clinical

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Clinical

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  Clinical Science Immediate Publication. Published on 23 Sep 2008 as manuscript CS20080392 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080392 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society history of a CAD. Regarding current medication of the patients, a trend for lower DCP levels was observed for statins, aspirin, and blockers. Different serum parameters such as

Clinical

  Science Immediate Publication. Published on 23 Sep 2008 as manuscript CS20080392 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080392A c c e p t e d M a n u s c r i p t creatinine > 1.04 mg/dL might be predictive of a CAD too, but failed to reach statistical significance. To analyze if the decrease in circulating DCP is a predictor of a CAD independent of the other variables shown in the univariate analysis, multiple stepwise logistic regression analysis was performed. Since circulating mDCP, pDCP, and tDCP showed a high correlation with each other, separate multiple stepwise logistic regression analyses were performed for their relative and absolute values in a model together with age, gender, diabetes mellitus, hypertension, HDL cholesterol, creatinine, and hsCRP (Fig.5B). In these analyses, reduction of circulating DCP either as relative (mDCP, pDCP, and tDCP) or absolute (pDCP and tDCP) values remained the only independent predictor of a CAD.

Clinical

  Science Immediate Publication. Published on 23 Sep 2008 as manuscript CS20080392 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080392

Clinical

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Clinical

  Science Immediate Publication. Published on 23 Sep 2008 as manuscript CS20080392 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080392

Clinical

  Science Immediate Publication. Published on 23 Sep 2008 as manuscript CS20080392 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080392 A c c e p t e d M a n u s c r i p t

Clinical

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Figure 1

 1 Figure 1 Representative dot plots showing the gating strategy used for the identification

Figure 2

 2 Figure 2 Frequency of circulating mDCP, pDCP, and tDCP in different stages of coro-

Figure 3

 3 Figure 3 Relative and absolute frequency of circulating mDCP, pDCP, and tDCP accord-

Figure 4

 4 Figure 4 Correlation of circulating DCP and other factors with the CAD Score. Upper

Figure 5

 5 Figure 5 Predictive value of the decrease in circulating DCP for the presence of a CAD.

  Figure 1

  Figure 5

  Clinical Science Immediate Publication. Published on 23 Sep 2008 as manuscript CS20080392 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080392

Table II Subgroup analysis of DCP values compared to clinical data or medication

 II Absolute numbers of mDCP, pDCP, and tDCP (cells / µL) are calculated for the presence (+) or absence (-) of a clinical feature or medication. Values are reported as median. Statistical analysis was performed using the Mann-Whitney rank sum test.

			mDCP			pDCP			tDCP
		-	+	P	-	+	P	-	+	P
	Age > 66 years	15.6	15.5	NS	8.9	7.9	NS	27.3	25.9	NS
	Male gender	15.0	15.5	NS	7.3	8.2	NS	23.9	25.7	NS
	Atherogenic risk factors (%)	x			x			X
	Diabetes mellitus	15.3	14.8	NS	8.0	7.0	NS	24.8	23.9	NS
	Hypertension	16.6	15.1	NS	9.5	7.3	0.001	27.4	24.3	0.01
	Smoking	14.8	14.8	NS	7.1	8.4	NS	17.2	19.1	NS
	Hyperlipidemia	16.1	15.1	NS	8.7	7.4	0.03	28.9	24.2	0.02
	Family history of CAD	15.3	15.6	NS	7.8	7.8	NS	25.1	25.6	NS
	Current Medication (%)
	Statins	18.1	17.8	NS	9.0	8.6	NS	29.1	27.9	NS
	Aspirin	15.7	15.1	NS	9.2	8.3	NS	27.3	25.3	NS
	Blocker	16.7	16.0	NS	8.5	8.3	NS	27.3	26.1	NS
	ACE inhibitors	15.0	15.4	NS	8.0	7.7	NS	25.9	24.7	NS
	Serum parameters
	LDL cholesterol > 130mg/dL	14.4	15.5	NS	7.4	8.3	NS	23.5	26.1	NS
	HDL cholesterol > 45 mg/dL	14.5	16.2	NS	7.2	8.3	NS	18.2	20.5	NS
	hsCRP > 2.0 mg/L	16.8	14.5	NS	8.0	7.9	NS	26.7	25.4	NS
	Creatinine >1.0 mg/dL	14.9	15.4	NS	7.7	7.9	NS	24.4	24.8	NS
	Leukocytes > 7300 cells/µL	13.9	16.8	0.002	6.9	8.5	0.002	23.2	28.2	< 0.001

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µL) pDCP (cells/µL) tDCP (cells/µL)

  Clinical Science Immediate Publication. Published on 23 Sep 2008 as manuscript CS20080392 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080392

	A. Univariate Analysis	B. Multiple Stepwise Logistic Regression Analysis
	Odds Ratio (95% CI), P						Odds Ratio (95% CI ), P
	1.74 (1.07-2.82), 0.02 1.57 (0.96-2.56), 0.07 1.74 (1.01-3.00), 0.04 1.76 (0.91-3.41), 0.09 0.56 (0.32-0.97), 0.03 1.53 (0.93-2.50), 0.09 1.87 (1.12-3.10), 0.01 0.38 (0.24-0.61), < 0.001 0.32 (0.20-0.53), < 0.001 0.29 (0.18-0.47), < 0.001 0.51 (0.32-0.83), < 0.001	Age Male Diabetes Hypertension HDL cholesterol Creatinine hsCRP mDCP (%) pDCP (%) tDCP (%) mDCP (cells/1.67 (0.80-3.50), 0.17 1.99 (0.86-4.61), 0.11 0.85 (0.37-1.96), 0.69 0.92 (0.35-2.41), 0.86 0.75 (0.37-1.51), 0.42 1.09 (0.51-2.33), 0.82 1.43 (0.70-2.94), 0.33 0.43 (0.21-0.85), 0.01 0.28 (0.14-0.56), < 0.001 0.28 (0.14-0.56), < 0.001 0.59 (0.30-1.17), 0.13
	0.38 (0.23-0.62), < 0.001						0.36 (0.18-0.71), 0.004
	0.39 (0.24-0.64), < 0.001						0.46 (0.23-0.92), 0.02
		0,1	0,2	0,5	1	2	5

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© 2008 The Authors Journal compilation © 2008 Biochemical Society


Adam Mullan
email: adamwfmullan@yahoo.co.uk

Naveed Sattar
email: nsattar@clinmed.gla.ac.uk

Dr Adam Mullan Or Professor

More knocks to the oxidation hypothesis for vascular disease?

The oxidation hypothesis for coronary heart disease (CHD) is attractive. However, the almost universal failure of antioxidant vitamin supplementation as a CVD risk modifier challenges the oxidation hypothesis, at least as a concept that easily 'translates' into clinical benefit for the population. At the same time, quality prospective data on lipid or protein oxidation markers as predictors of vascular events is spare. In this issue of the journal, Woodward and colleagues provide much needed prospective data examining the relationship between markers of oxidative damage and CHD outcome in a general population. Despite noting the expected associations between several established CHD risk factors and CHD events, no significant link was seen between measured oxidation markers and CHD risk, a finding which further challenges the oxidation hypothesis for CHD.

Healthy human physiology relies on a complex balance of pro-and antioxidant mechanisms. Reactive oxygen species such as superoxide anions, free radicals and hydrogen peroxide, are generated as by-products of aerobic metabolism. These prooxidant systems are balanced by a battery of antioxidants such as exogenously derived ascorbic acid and endogenous superoxide dismutase. Pro-oxidant effects are essential for normal processes involved in cell apoptosis and innate immune responses to infection. When oxidative stress occurs in excess and overwhelms anti-oxidant reserves, lipids, proteins and DNA are indiscriminately damaged, often rendering them dysfunctional and contributing to the development of chronic disease.

A central role for oxidative stress in cardiovascular disease (CVD) is an attractive theory, particularly given that antioxidant administration (in the form of 'simple and safe' vitamin supplements) might reverse or at least slow this process in some way. Prior consistent epidemiological evidence of low vitamin levels in subjects at risk of CVD has fuelled an explosion of end-point trials investigating the cardio-protective potential of vitamin supplements. Antioxidant supplementation has, however, generally failed to demonstrate any beneficial effect. A recent Cochrane meta-analysis of 77 randomised trials involving 232,550 participants concluded that there was no evidence to support the use of antioxidants in primary or secondary CVD prevention and, in fact, suggested a potentially deleterious effect with vitamin A, beta-carotene and Vitamin E [START_REF] Bjelakovic | Antioxidant supplements for prevention of mortality in healthy participants and patients with various diseases[END_REF]. Similarly, observational studies have linked circulating homocysteine levels with the risk of CVD; however despite successfully lowering homocysteine levels, interventional studies of folic acid and cyanocobalamin have not shown any reduction in CVD risk [2;3]. This illustrates an important concept: attempting to reverse the pathophysiological mechanisms linked to apparently aberrant biomarker levels (in this case low antioxidant levels) does not necessarily translate into improved outcomes.

The almost universal failure of antioxidant vitamin supplementation as a CVD risk modifier certainly threatens the oxidation hypothesis, at least as a concept that translates into clinical benefit for the population. However, a number of considerations have been proposed in this area to try to explain past failures and to help improve future research. The Cochrane review suggests that trial methodologies in the antioxidant field have generally been suboptimal: patient groups varied dramatically with no estimation of baseline oxidative stress levels; there was uncertainty regarding baseline and cardiovascular health at outcome; and risk of bias was high. The interventional supplements also varied massively in terms of dose, duration of treatment, and combinations used. Of course, the results of the antioxidant randomised controlled trials (RCTs) have to be taken at face value; there is currently no role for supplements of naturally occurring antioxidants in the prevention of CVD. With this in mind, it is of interest that Lawlor and colleagues recently showed that socio-economic factors may confound the relationship between low vitamin levels and CVD risk [START_REF] Lawlor | Those confounded vitamins: what can we learn from the differences between observational versus randomised trial evidence?[END_REF]. In other words, low vitamin levels may not be causally linked to CVD risk; rather they are simply markers of some other more relevant process also linked to socio-economic status.

With the above body of evidence in mind, there is clearly a need for parallel prospective studies linking oxidative stress markers measured at baseline to incident CVD outcomes. But which markers are best and how easy are they to measure? An ever increasing number of biomarkers of oxidative stress are emerging, relying on identification of invivo lipid and protein targets for oxidation (reviewed [5;6]). F2-Isoprostanes, derived from non-enzymatic free-radical mediated peroxidation of arachidoic acid, are arguably the best established biomarker of oxidative stress. In relation to CVD, they have been found to be significantly increased in atherosclerotic plaques compared to healthy nonatherosclerotic vascular tissue; systemic F2-Isoprostanes have also been found to increase following angioplasty. The relationship between oxidized LDL-cholesterol (oxLDL) and CVD has also been extensively studied, and there is some evidence, albeit mixed, for oxLDL markers as in-vivo markers of oxidative stress. Unregulated uptake of oxLDL by scavenger receptors on macrophages results in the formation of foam cells, precursors of advanced atheromatous plaque formation. OxLDL is also thought to i) enhance interactions between monocytes and endothelial cells to promote atherogenesis and plaque instability, ii) induce the renin-angiotensin system, and iii) promote a cascade of inflammatory cytokines such as IL-6, and TNF-alpha. Finally, HDL particles are another target for oxidative stress, and it is possible to measure oxidised apolipoproteins (apo) A-I and A-II as markers for in-vivo HDL-cholesterol oxidation. The role of oxidised HDL (oxHDL) in atherogeneis is however less clear. Studies have suggested that oxidation of methionine residues on apoA-I impairs reverse cholesterol transport; however studies in mice suggest that tyrosyl radical oxHDL may be more effective at extracting cholesterol [START_REF] Macdonald | Administration of tyrosyl radical-oxidized HDL inhibits the development of atherosclerosis in apolipoprotein E-deficient mice[END_REF].

Measurement of markers of oxidative stress is therefore complex and also requires careful sample collection and storage, and invariably labour intensive methodology, such as high pressure liquid chromatography (HPLC) for oxHDL or mass spectrometry for F2-isoprostanes, as described by Woodward et al [START_REF] Woodward | The association between both lipid and protein oxidation and the risk of fatal or non-fatal coronary heart disease in a human population[END_REF]. Other methods involve immunoassays to oxidised moieties of oxLDL. However there are lingering concerns over specificity of some ELISA based methods as well as uncertainty about the pathophysiological relevance of some oxidative markers measured in the circulation versus tissue levels (where levels may change in opposite directions). Accurate assessment or interpretation of oxidative stress markers in large scale population based studies is therefore not easy and, as a result, there are few robust studies prospectively linking oxidative biomarkers with CVD incident end-points. Small-scale studies have suggested a role for F2-Isoprostanes (measured in plasma or urine) as independent risk markers for atherosclerosis [9;10]; however larger prospective studies, with proper adjustment for confounding factors, are noticeably sparse [START_REF] Tsimikas | C-reactive protein and other emerging blood biomarkers to optimize risk stratification of vulnerable patients[END_REF].

In the current issue of the journal, Woodward et. al report a new prospective study in this area. They measured F2-Isoprostanes, and oxidised moieties on apoliprotein AI and AII, HDL-related proteins, in a nested case control study with 227 coronary cases and 420 controls. Woodward et al. noted no significant association between measured markers and CVD event risk. Whether this study had sub-optimal power to detect associations between oxidation markers and CHD risk is debatable since established risk predictors were significantly related to outcomes. In short, the results are disappointing with regards to the oxidation hypothesis. There is little other data on oxHDL moieties available for comparison but other markers of oxidations, for example oxidised phospholipids /apoB measurement (an antibody based assay) has been shown by others to be predictive of 10year CVD events in a prospective population based study of 765 subjects [START_REF] Kiechl | Oxidized phospholipids, lipoprotein(a), lipoprotein-associated phospholipase A2 activity, and 10-year cardiovascular outcomes: prospective results from the Bruneck study[END_REF]. To advance this field, careful collaboration between different groups is required to generate greater power in prospective studies of a range of oxidative markers. Such studies should also employ, wherever possible, gold standard or at least standardised laboratory methods to measure oxidative stress so that results are more robust. In summary, although the oxidation hypothesis for vascular disease is attractive, there is currently inconsistent evidence linking markers of oxidative stress with incident CVD events. Equally, RCTs of vitamins have failed to show vascular benefit and in some cases have suggested possible harm. That said, a recent RCT with a synthetic antioxidant, succinobucol [12], did show some promise in terms of vascular and diabetes risk reduction although the trial failed to meet its primary end-point and reported some unexpected harms. Given these uncertainties, the foundations of CVD risk prediction should continue with established risk factors such as lipids, blood pressure, and smoking to determine risk and proven interventions to lessen it.

Clinical

  Science Immediate Publication. Published on 10 Oct 2008 as manuscript CS20080486 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080486 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 10 Oct 2008 as manuscript CS20080486 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080486 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 10 Oct 2008 as manuscript CS20080486 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080486 A c c e p t e d M a n u s c r i p t

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MD Stephan Von Haehling
email: stephan.von.haehling@web.de

STATINS FOR HEART FAILURE: STILL CAUGHT IN NO MAN'S LAND?

Statins are well-known for their ability to lower serum cholesterol levels, but have properties beyond mere cholesterol reduction. These pleiotropic effects include an improvement in endothelial dysfunction, release of endothelial progenitor cells, anti-inflammatory properties, and a number of antitumour activities. In the present issue of Clinical Science, Stunpf et al. show that a 4-week treatmentcourse with the lipophilic atorvastatin ameliorates left ventricular remodelling and function, reduces serum levels of TNFα, IL-6, and monocyte chemoattractant protein-1, and increases both serum and myocardial levels of IL-10. The authors hypothesize that this shift from a pro-to an anti-inflammatory response might be beneficial in the clinical setting, because patients with low levels of IL-10 may fare worse than those with higher levels. This notion requires further investigation, but highlights the need to identify those patients who are likely to benefit from statin therapy. Statins can lower serum cholesterol levels. That fact is beyond doubt. Patients with or at high risk of atherosclerosis benefit from statin treatment: another fact

that is well buttressed by a large number of studies in both primary and secondary prevention. However, whether or not cholesterol reduction with statins is an aim worth pursuing in certain chronic conditions is an entirely different matter, and particularly the concept of cholesterol reduction in patients with heart failure (HF) has recently been called into question [START_REF] Von Haehling | Statins for heart failure: at the crossroads between cholesterol reduction and pleiotropism?[END_REF].

The good news about statins is that they possess properties beyond mere cholesterol reduction. These pleiotropic effects include an improvement in endothelial dysfunction [START_REF] Von Haehling | Statins and the role of nitric oxide in chronic heart failure[END_REF], release of endothelial progenitor cells, antiinflammatory properties, and a number of anti-tumour activities [START_REF] Wong | Cerivastatin triggers tumor-specific apoptosis with higher efficacy than lovastatin[END_REF][START_REF] Jakobisiak | Cell cycle-specific effects of lovastatin[END_REF]. The improvement in endothelial function mediated by atorvastatin, for example, which has been demonstrated in an elegant animal model of HF post myocardial infarction, is probably due to an increase in nitric oxide production and a reduction in its inactivation [START_REF] Landmesser | Statin-induced improvement of endothelial progenitor cell mobilization, myocardial neovascularization, left ventricular function, and survival after experimental myocardial infarction requires endothelial nitric oxide synthase[END_REF]. Atrovastatin at a dose of 10 mg once daily for 4 weeks also achieved an improvement in forearm vasodilatory response to reactive hyperaemia in HF patients [START_REF] Tousoulis | Effects of atorvastatin on reactive hyperemia and inflammatory process in patients with congestive heart failure[END_REF].

A large number of studies have shown statins' beneficial effects with regards to markers of inflammation including C-reactive protein, tumour necrosis factor-α (TNFα), and interleukin (IL) 1 and 6 [7--10]. Overactivity of the immune system has been a matter of ongoing concern in HF patients for almost two decades now [START_REF] Anker | Inflammatory mediators in chronic heart failure: an overview[END_REF], and especially TNFα and its soluble receptors have been demonstrated to be markers of an adverse prognosis in patients with this disease [START_REF] Rauchhaus | Plasma cytokine parameters and mortality in patients with chronic heart failure[END_REF][START_REF] Ferrari | Tumor necrosis factor soluble receptors in patients with various degrees of congestive heart failure[END_REF]. However, large-scale studies to directly antagonize this cytokine with specific antibodies involving more than 2000 patients ended in disappointment [START_REF] Mann | Targeted anticytokine therapy in patients with chronic heart failure: results of the Randomized Etanercept Worldwide Evaluation (RENEWAL)[END_REF][START_REF] Chung | Anti-TNF Therapy Against Congestive Heart Failure Investigators. Randomized, double-blind, placebo-controlled, pilot trial of infliximab, a chimeric monoclonal antibody to tumor necrosis factor-alpha, in patients with moderate-to-severe heart failure: results of the anti-TNF Therapy Against Congestive Heart Failure (ATTACH) trial[END_REF]. It therefore appears the right time to choose novel weapons in the battle against HF, and the overactivity of the immune system is still an interesting target [START_REF] Von Haehling | Future prospects of anticytokine therapy in chronic heart failure[END_REF].

As statins possess anti-inflammatory properties, the publication of prospective large-scale studies of these drugs in patients with HF were eagerly awaited. The CORONA (Controlled Rosuvastatin Multinational Trial in Heart Failure) study was a double-blind study that enrolled 5,011 patients with ischaemic disease and an impaired left ventricular ejection fraction who were randomized to placebo or rosuvastatin at a dose of 10 mg once daily [START_REF] Kjekshus | Rosuvastatin in Older Patients with Systolic Heart Failure[END_REF]. Patients were treated for a median follow-up of 32.8 months. Unfortunately, no significant difference in the primary end-point, a composite of death from cardiovascular causes, non-fatal myocardial infarction, or non-fatal stroke, was noted between the rosuvastatin and the placebo group (hazard ratio [HR] 0.92, 95% confidence interval [CI] 0.83-1.02, p=0.12). The prospective, multicenter, randomized, double-blind GISSI-HF trial (Gruppo Italiano per lo Studio della Sopravvivenza nell'Insufficienza Cardiaca) was published in late August 2008 [START_REF] Investigators | Effect of rosuvastatin in patients with chronic heart failure (the GISSI-HF trial): a randomised, double-blind, placebo-controlled trial[END_REF]. For this study, 4,574 patients were randomized to the same regimen with rosuvastatin 10 mg once daily or placebo. Like with CORONA, there was no difference in the primary endpoints of time to death (HR 1.00, 95% CI 0.90-1.12, p=0.94) and time to death or hospitalization for cardiovascular reasons (HR 1.01, 95% CI 0.91-1.11, p=0.90) between the two study arms. The publication of these two studies was a serious blow to the "statins-in-heartfailure" story. An earlier, yet less heeded setback were the results of a prospective study that was initially named the UNIVERSE (Rosuvastatin Impact on Ventricular Remodeling, Cytokines and Neurohormones) trial, in which 86 patients were randomized to placebo or rosuvastatin at an increasing dose (target: 40 mg once daily) in a double-blind fashion [START_REF] Krum | Double-blind, randomized, placebo-controlled study of high-dose HMG CoA reductase inhibitor therapy on ventricular remodeling, pro-inflammatory cytokines and neurohormonal parameters in patients with chronic systolic heart failure[END_REF]. Six months of treatment did not yield any improvement in the plasma levels of C-reactive protein, TNFα, IL-6, or B-type natriuretic peptide. Additionally, there was no significant improvement in left ventricular ejection fraction.

One may wonder if these studies should set an end to studying statins in HF. However, as Sir Arthur Conan Doyle pointed out, "it is a capital mistake to theorize before you have all the evidence. It biases the judgement [START_REF] Ratcliffe | The Oxford dictionary of quotations by subject[END_REF]." Thus, one may alternatively wonder if rosuvastatin was simply the wrong statin. Or used at a wrong dose. Indeed, rosuvastatin is one of the very few hydrophilic statins [START_REF] Von Haehling | Statins[END_REF], and it has been suggested that lipophilic statins might be able to enter biological membranes more easily which yields more pleiotropic effects [START_REF] Liao | Pleiotropic effects of statins[END_REF]. On the other hand, it is intriguing to note that higher cholesterol levels are associated with better (not worse) survival in patients with HF in several retrospective analyses [START_REF] Horwich | Low serum total cholesterol is associated with marked increase in mortality in advanced heart failure[END_REF][START_REF] Rauchhaus | The relationship between cholesterol and survival in patients with chronic heart failure[END_REF]. A potential explanation is that cholesterolcontaining lipoproteins can detoxify the bacterial cell-wall component endotoxin [START_REF] Rauchhaus | The endotoxin-lipoprotein hypothesis[END_REF][START_REF] Von Haehling | The cholesterol paradox revisited: heart failure, systemic inflammation, and beyond[END_REF], which appears to play a significant role in patients with HF during oedematous decompensation [START_REF] Anker | Elevated soluble CD14 receptors and altered cytokines in chronic heart failure[END_REF], but it may also be responsible for immune activation in these patients under stable conditions [xi]. The available data should therefore set the stage to undertake studies in HF that fulfill two criteria: (i) to investigate statins at doses that DO NOT lower cholesterol values (but confer pleiotropic effects) and (ii) to use statins other than rosuvastatin and potentially pravastatin, the other hydrophilic substance.

The study by Stumpf et al. in this issue of the Journal comes therefore at the right time [START_REF] Stumpf | Atorvastatin enhances interleukin-10 levels and improves cardiac function in rats after acute myocardial infarction[END_REF]. Although the authors do not provide data on cholesterol, they show that a 4-week treatment-course with the lipophilic atorvastatin ameliorates left ventricular remodelling and function, reduces serum levels of TNFα, IL-6, and monocyte chemoattractant protein-1, and increases both serum and myocardial levels of IL-10. The authors hypothesize that this shift from a pro-to an antiinflammatory response might be beneficial in the clinical setting, because patients with low levels of IL-10 may fare worse than those with higher levels, as suggested by several studies [START_REF] Girndt | Interleukin-10 (IL-10)[END_REF]. This notion highlights another important fact: We need to identify those patients who are likely to benefit from statin therapy. This was not even an inclusion criterion for the studies of antibody therapy against TNFα [30]. It could well be that the levels of pro-or anti-inflammatory markers could help in that sense.

Clinical

  Science Immediate Publication. Published on 21 Oct 2008 as manuscript CS20080530 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080530 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 21 Oct 2008 as manuscript CS20080530 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080530 A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t

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T J Corte

Dr S J Wort
email: s.wort@imperial.ac.uk

The Role of Pravastatin in Pulmonary Hypertension in Chronic Obstructive Pulmonary Disease

In this issue of Clinical Science Lee et al report a placebo-controlled study of 53 patients with chronic obstructive pulmonary disease (COPD) and pulmonary hypertension (PH) on echocardiography, in which six-months of pravastatin treatment was associated with a significant increase in exercise time (660 to 1006s, p<0.0001) and improvement in Borg dyspnoea score. COPD is clearly a significant health concern as the fourth leading cause of morbidity and mortality in the US, costing up to £1 billion per year in the UK. The exact prevalence of PH in COPD is uncertain. However, it is estimated that mean pulmonary artery pressure will rise on average by 0.28 to 0.6 mmHg per year per patient, so occurring in up to 95% of patients awaiting lung volume reduction surgery or transplantation. However, PH in this context is usually mild to modest in severity, except for a small number with COPD 'out-of-proportion' to their underlying airflow limitation. Certainly, the presence of PH is a risk factor for mortality in patients with COPD. We review the role of PH in COPD and its pathophysiology, with reference to endothelin-1, cigarette smoke, as well as exercise and nocturnal hypoxia. We also explore potential mechanisms for the observed improvement in exercise tolerance seen in six months' pravastatin treatment, including possible effects upon endothelin-1, Rho-kinase, or antioxidant effects, which may be particularly relevant in this group of mainly current smokers.

In this issue of Clinical Science Lee et al report a placebo-controlled study of 53 patients with chronic obstructive pulmonary disease (COPD) and pulmonary hypertension (PH) on echocardiography, in which six-months of pravastatin treatment was associated with a significant increase in exercise time (660 to 1006s, p<0.0001) and improvement in Borg dyspnoea score [START_REF] Lee | Effects of pravastatin on functional capacity in patients with chronic obstructive pulmonary disease and pulmonary hypertension[END_REF] .

COPD is clearly a significant health concern as the fourth leading cause of morbidity and mortality in the US, costing up to £1 billion per year in the UK. But how important is the association of PH with this condition? The exact prevalence of PH in COPD is uncertain. However, it is estimated that mean pulmonary artery (PA) pressure will rise on average by 0.28 to 0.6 mmHg per year per patient [START_REF] Kessler | Natural history" of pulmonary hypertension in a series of 131 patients with chronic obstructive lung disease[END_REF][START_REF] Weitzenblum | Evolution of pulmonary hypertension in chronic obstructive pulmonary diseases[END_REF] . The outcome of this is that it is likely that a significant proportion of patients will develop PH over their lifetime, with reports extending up to 95% in patients awaiting lung volume reduction surgery or transplantation [START_REF] Scharf | Hemodynamic characterization of patients with severe emphysema[END_REF][START_REF] Vizza | Right and left ventricular dysfunction in patients with severe pulmonary disease[END_REF][START_REF] Chaouat | Severe pulmonary hypertension and chronic obstructive pulmonary disease[END_REF] . However, PH in this context is usually mild to modest in severity. The exception to this is the group of 'outliers' with more significant PH 'out-of-proportion' to the underlying airflow limitation [START_REF] Chaouat | Severe pulmonary hypertension and chronic obstructive pulmonary disease[END_REF] . Importantly, the presence of PH does confer a worse prognosis, being an independent factor for both mortality and frequency of COPD exacerbations [START_REF] Oswald-Mammosser | Prognostic factors in COPD patients receiving long-term oxygen therapy. Importance of pulmonary artery pressure[END_REF][START_REF] Kessler | Predictive factors of hospitalization for acute exacerbation in a series of 64 patients with chronic obstructive pulmonary disease[END_REF] .

PH in the context of COPD, is classified as PH related to hypoxia and/or chronic respiratory disease [START_REF] Simonneau | Clinical classification of pulmonary hypertension[END_REF][START_REF] Simonneau | Proceedings of the 3rd World Symposium on Pulmonary Arterial Hypertension[END_REF] . However, is it reasonable to consider PH in COPD as primarily hypoxia-related? Traditionally, PH in COPD was thought to be a related to destruction of the vascular bed which occurs in emphysema, and as a response to chronic hypoxia. However, PH is not associated with the degree of emphysema 4 , occurs independent of hypoxia and is not completely ameliorated by oxygen replacement [START_REF] Oswald-Mammosser | Prognostic factors in COPD patients receiving long-term oxygen therapy. Importance of pulmonary artery pressure[END_REF] . Other mechanisms, must, therefore, be considered in the pathophysiology of PH, particularly when 'out-ofproportion' to the underlying airflow limitation. The role of transient hypoxia and PH seen on exercise, at night, and during COPD exacerbations, is yet to be determined in the pathophysiology of resting PH [START_REF] Kessler | Natural history" of pulmonary hypertension in a series of 131 patients with chronic obstructive lung disease[END_REF][START_REF] Fletcher | Pulmonary vascular hemodynamics in chronic lung disease patients with and without oxyhemoglobin desaturation during sleep[END_REF][START_REF] Abraham | Factors contributing to the reversible pulmonary hypertension of patients with acute respiratory failure studies by serial observations during recovery[END_REF] . It is also possible that cigarette smoke, itself, may play a role in endothelial dysfunction, and subsequent vascular damage [START_REF] Wright | Pulmonary hypertension in chronic obstructive pulmonary disease: current theories of pathogenesis and their implications for treatment[END_REF][START_REF] Wright | Cigarette smoke induces persisting increases of vasoactive mediators in pulmonary arteries[END_REF] . Currently, apart from oxygen therapy, there is no treatment for PH associated with COPD.

Endothelin-1 (ET-1) is a vasoactive mediator that causes vasoconstriction and vascular cell proliferation. In idiopathic pulmonary arterial hypertension (PAH) levels of ET-1 in plasma correlate to pulmonary vascular resistance and inversely to prognosis [START_REF] Stewart | Increased plasma endothelin-1 in pulmonary hypertension: marker or mediator of disease?[END_REF][START_REF] Galie | Relation of endothelin-1 to survival in patients with primary pulmonary hypertension[END_REF] . Blockade of the ET-1 A and B receptors with a mixed receptor blocker, bosentan, improves functional status and overall mortality. There is gathering evidence that blockade of the ET-1 system may also benefit patients with PAH in association with Eisenmenger syndrome, connective tissue disease, HIV and thromboembolic disease. ET-1 also has potent bronchoconstrictive properties and has been implicated in the pathogenesis of airways disease. The role of ET-1 in the pathophysiology of COPD remains uncertain, and may be related to its effect upon bronchoconstriction, or its proinflammatory properties. Plasma ET-1 levels are elevated in COPD patients compared to control subjects [START_REF] Fujii | Plasma endothelin-1 level in chronic obstructive pulmonary disease: relationship with natriuretic peptide[END_REF] A c c e p t e d M a n u s c r i p t higher day and night ET-1 plasma levels [START_REF] Nikolaou | Evaluation of arterial endothelin-1 levels, before and during a sleep study, in patients with bronchial asthma and chronic obstructive pulmonary disease[END_REF] . It is possible that ET-1 may contribute to the development of PH in COPD. However, reports of ET-1 levels in PH in COPD have been conflicting; with one study showing no difference in levels amongst patients with or without PH [START_REF] Bacakoglu | Plasma and bronchoalveolar lavage fluid levels of endothelin-1 in patients with chronic obstructive pulmonary disease and pulmonary hypertension[END_REF] , and another study demonstrating increased exhaled and arterial ET-1 levels in patients with PH (with ET-1 levels correlating with echocardiographic systolic pulmonary arterial pressures) [START_REF] Carratu | Exhaled and arterial levels of endothelin-1 are increased and correlate with pulmonary systolic pressure in COPD with pulmonary hypertension[END_REF] . There has been one randomised study investigating the effect of bosentan (versus placebo) on cardiopulmonary hemodynamics during exercise in patients with severe COPD, but importantly, with no resting PH. There was no improvement in six-minute walk test, the primary outcome measure. In addition, there was deterioration in oxygenation and quality of life with no change in pulmonary artery pressure [START_REF] Stolz | A randomised, controlled trial of bosentan in severe COPD[END_REF] .

Statins, the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, including pravastatin, block the rate-limiting step in cholesterol synthesis. However, their benefit clearly extends beyond the effect upon lowering cholesterol. Statins have 'pleiotropic' effects including the improvement of endothelial dysfunction, inhibition of vascular smooth muscle proliferation, and induction of vascular smooth muscle cell apoptosis. The mechanisms by which these effects is uncertain, but appears to be related to the inhibition of synthesis of small GTP binding proteins required for the RhoA activation. This, in turn, leads to the reduction in endothelial nitric oxide synthase (eNOS) and decreased nitric oxide production and subsequent vasoconstriction. Moreover, RhoA/Rho-kinase signalling plays an important role in the sustained phase of vasoconstriction mediated by ET-1. While statins prevent and ameliorate PH in animal studies, there is no data for their use in the treatment of PH from any cause.

In this context, the study by Lee et al reported in this Journal, sets out to test the hypothesis that statins may improve hemodynamics in patients with COPD and PH, by affecting ET-1 synthesis. This was based on a previous observation that statins inhibit prepro ET-1 transcription in vitro. 53 patients with stable COPD (FEV1 56% predicted) and PH (defined as systolic PA pressure greater than 35 mmHg by echocardiography) were randomised to receive either pravastatin or placebo in a double-blind manner over a period of 6 months. Exercise capacity, tested using a Naughton stress-test treadmill protocol, was significantly improved in the pravastatin treated group and remained static in the placebo group. Systolic PA pressure significantly fell from 47 mmHg to 40 mmHg and Borg dyspnoeic index also improved. There was no change in plasma ET-1 measured in peripheral vein but a slight decrease in urinary excretion of ET-1. There was a significant correlation between changes in exercise time and levels of ET-1 excreted in the urine. The main conclusion of the authors was that "pravastatin significantly improved exercise tolerance, decreased PH, and dyspnoea during exercise, probably by inhibiting endothelin-1 synthesis in COPD patients with PH". So where does this leave us with the story of COPD, PH and ET-1? The most impressive finding of the study by Lee et al was the improvement in exercise capacity in patients receiving pravastatin. Was this due to an improvement in PH? Of course, this study was not designed to answer this question. We note the parallel study published by the same authors, in which a similar benefit was demonstrated across COPD patients independent of the presence of PH [START_REF] Lee | Usefulness of C-reactive protein and interleukin-6 as predictors of outcomes in patients with chronic obstructive pulmonary disease receiving pravastatin[END_REF] . Also, given the modest elevation in systolic PA pressure in patients at the beginning of the study, it is unlikely that PH was the limiting factor and therefore that the improvement in PH on treatment was the reason for improved exercise capacity. The caveat to this statement is that we do not know if pravastatin (acutely and with time) ameliorates the transient elevation in PA pressure seen on exercise, which is a known risk factor for the development of resting PH in COPD [START_REF] Oswald-Mammosser | Prognostic factors in COPD patients receiving long-term oxygen therapy. Importance of pulmonary artery pressure[END_REF] . Also, as the authors note, it is difficult to assess the therapeutic benefit with regard to PH, in the absence of the resting, exercise and nocturnal oxygen levels.

What about the role of ET-1? In the reported study, there was no change in venous plasma ET-1 with treatment. As most ET-1 is produced in the lung, perhaps a better measure would have been trans-pulmonary ET-1 production (i.e. comparisons of central venous and arterial samples). However, a reduction in renal excretion of ET-1 was observed, although, as the authors stated, the magnitude was not enough to explain the change in exercise capacity (R 2 = 0.22). It is difficult to propose how a reduction in renal excretion of ET-1 would account for the improved exercise capacity. The authors note that urinary ET-1 excretion correlates with global ET-1 levels, and may be more sensitive to changes in overall endogenous ET-1 production than serum ET-1 levels.

However, the beneficial effect of pravastatin is more likely to be independent of ET-1. The authors mention two possible alternative mechanisms in which pravastatin may affect vascular function: oxidative stress and the RHO-kinase system. Oxidative stress is increased in COPD, and is obviously important in cigarette induced vascular injury. Statins may reduce free radical generation. In this cohort of mainly (81%) current smokers, any antioxidant mechanisms may have been particularly effective. It would be interesting to determine if a similar benefit is seen in ex or never smokers. Statins are also known to effect apoptosis of vascular cells, which may have a longer-term effect on vascular remodelling.

In summary, the most exciting finding from the study of Lee et al is the beneficial effect of pravastatin on exercise capacity in patients with COPD and PH. The exact mechanism of this benefit is uncertain, as is the contribution of PH. The role of ET-1 in PH in association with COPD is also unclear and some doubt has already been cast on the usefulness of ET-1 receptor antagonists in this condition. Clearly, more work needs to be done on the contribution of exercise-induced increases in PH in COPD and the effect of this upon exercise capacity. In addition, we need to study the importance of ET-1 and blocking its action in patients with COPD and moderate to severe PH, although this appear to be a minority group.

  . Plasma ET-1 increases during acute exacerbations, and falls following one hour of oxygen replacement. Patients with COPD with nocturnal desaturation have Clinical Science Immediate Publication. Published on 27 Nov 2008 as manuscript CS20080590 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080590

Clinical

  Science Immediate Publication. Published on 27 Nov 2008 as manuscript CS20080590 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080590 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 27 Nov 2008 as manuscript CS20080590 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080590 A c c e p t e d M a n u s c r i p t

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© 2008 The Authors Journal compilation © 2008 Portland Press Limited