Effects of levosimendan on flow-mediated vasodilation and soluble adhesion molecules in patients with advanced chronic heart failure

Atherosclerosis 197 (2008) 278–282

Effects of levosimendan on flow-mediated vasodilation and soluble adhesion molecules in patients with advanced chronic heart failure John T. Parissis a,∗ , Apostolos Karavidas b , Vassiliki Bistola a , Sophia Arapi b , Ioannis A. Paraskevaidis a , Dimitrios Farmakis a , Dimitrios Korres b , Gerasimos Filippatos a , Evaggelos Matsakas b , Dimitrios T. Kremastinos a a

Second Department of Cardiology, Attikon University Hospital, Rimini 1, 12461 Chaidari, Athens, Greece b Department of Cardiology, Geniko Kratiko Hospital, Athens, Greece Received 12 February 2007; received in revised form 20 April 2007; accepted 20 April 2007 Available online 12 June 2007

Abstract Aim: Endothelial activation and dysfunction may be an important contributor to chronic heart failure (CHF) progression. We sought to investigate whether the calcium sensitizer levosimendan affects beneficially endothelial function and attenuates the deleterious effects of soluble adhesion molecules in patients with advanced CHF. Methods: Twenty-six advanced CHF patients (mean New York Heart Association class, 2.6 ± 0.3; ischemic/dilated, 18/8; mean left ventricular ejection fraction <35%) hospitalized due to syndrome worsening, were randomized (2:1) to receive either a 24-h levosimendan infusion of 0.1 ␮g/kg/min (n = 17) or placebo (n = 9). Endothelial function estimated by endothelial-dependent flow-mediated dilatation of the brachial artery (FMD), as well as plasma soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM1), were assessed before and 48 h after therapy. Results: Baseline characteristics and medications were well balanced in the two treatment groups. A significant improvement of FMD (6.4 ± 4.4% from 4.8 ± 3.0%; p < 0.05) with concomitant reduction of plasma concentrations of sICAM-1 (231 ± 75 pg/ml from 339 ± 157 pg/ml; p < 0.05) and sVCAM-1 (1134 ± 508 pg/ml from 1386 ± 602 pg/ml; p < 0.05) were observed only in levosimendan treated patients. Conclusion: Levosimendan could be an effective treatment in improving the endothelial function and reducing the detrimental adhesion molecule activation in advanced CHF patients. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Levosimendan; Endothelial dysfunction; Flow-mediated vasodilation; Adhesion molecules; Advanced heart failure

1. Introduction Chronic heart failure (CHF) is associated with endothelial dysfunction, which contributes significantly to the pathophysiology and progression of the syndrome [1–3]. Several studies have shown that impaired endothelium-dependent flow-mediated vasodilation (FMD), mirroring endothelial dysfunction, represents an independent adverse prognostic

∗

Corresponding author. Tel.: +30 210 5831235; fax: +30 210 5832351. E-mail address: [email protected] (J.T. Parissis).

0021-9150/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2007.04.023

marker in CHF patients, being independently associated with cardiac death and hospitalization [4]. Levosimendan is a calcium-sensitizing inotropic agent, used for the short-term management of decompensated heart failure patients [5,6]. Parallel to its inotropic properties, levosimendan has been shown to be a potent vasodilator of the coronary, splachnic and pulmonary arterial vasculature [7–9], through the activation of ATP-regulated potassium channels located on the membranes of vascular arterial smooth muscle cells [10]. Recently, experimental data suggests that levosimendan also causes coronary vasodilation through the stimulation of nitric oxide (NO) release in coronary arteries

J.T. Parissis et al. / Atherosclerosis 197 (2008) 278–282

[11]. However, no in vivo data exists regarding the effects of levosimendan on endothelial function and vascular adhesion properties in patients with advanced CHF. In this placebo-controlled randomized trial, we aimed to investigate the effects of levosimendan on endothelialdependent vasodilatation in patients with advanced CHF, with parallel determination of plasma markers of neuro-hormonal (B-type natriuretic peptide, BNP), systemic inflammatory (interleukin-6, IL-6) and vascular inflammatory activation (sICAM-1, sVCAM-1).

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to 2 min after cuff deflation, and for 5 min after nitroglycerin administration. The baseline and maximum FMD and NMD diameters were measured from one media-adventitia interface to the other and calculated as the mean of five measurements over a short segment of the artery at end-diastole of a single cardiac cycle. Vasodilation was estimated as the percent change in diameter over the baseline value. Brachial artery blood flow velocity (cm/s) was determined with a 1.2 mm, pulsed-Doppler sampling volume in the vessel lumen midline with software correction for the incident angle of 60◦ at baseline and for the first 30 s after cuff release. The mean blood flow velocity over five cardiac cycles is reported.

2. Methods 2.3. Laboratory tests 2.1. Study population The study population consisted of 26 consecutive patients who were hospitalized for symptoms of heart failure and had a documented left ventricular ejection fraction <35%. Exclusion criteria were any acute or chronic infectious or inflammatory diseases, recent myocardial infarction (<8 weeks) or active myocardial ischemia, hepatic or renal impairment (creatinine > 2.5 mg/dl), use of antiinflammatory agents, serious arrhythmias and supine systolic blood pressure <85 mmHg. The investigation conformed to the principles outlined in the Declaration of Helsinki. All patients enrolled gave written informed consent. Patients were randomized (2:1) immediately after their admission and after optimization of oral medications to receive a 24-h infusion of either levosimendan (n = 17) or placebo (dextrose 5%; n = 9). Levosimendan was given at a rate of 0.1 ␮g/kg/min, without a loading dose. Patients of both study groups were evaluated by FMD and endothelium-independent nitroglycerin-mediated vasodilation (NMD) assessment and measurement of plasma levels of BNP, IL-6, sICAM-1 and sVCAM-1, before and 48 h after completion of levosimendan or placebo treatment. Thereafter, treatment was modified according to patients’ response. All ultrasound and biochemical tests were conducted by staff that was blinded to the treatment status of individual patients.

Venous blood samples were collected and centrifuged at 3500 rpm for 10 min at 4 ◦ C. Plasma aliquots were stored at −70 ◦ C for subsequent analyses. Plasma BNP was measured using the rapid Triage BNP assay (Biosite Inc., San Diego, California, USA). Circulating cytokines and soluble adhesion molecules ICAM and VCAM were measured using enzyme-linked immunosorbent assay kits (R&D Systems Inc., Minneapolis, MN, USA). 2.4. Statistical analysis Statistical analysis was performed using SPSS statistical analysis software (version 13.0). Categorical variables were compared using the Chi-square test. Mean values of continuous variables were compared between groups using the Student’s t-test or the Mann–Whitney U-test, according to whether variables were normally distributed or not, as tested by the Kolmogorov–Smirnov test. Similarly, the paired t-test or Wilcoxon’s paired test were used, respectively, to compare mean values before and after the therapeutic intervention (levosimendan or placebo). Assuming a levosimendan-induced increase in FMD of 2%, derived by preliminary data, a sample of 17 patients would detect this difference with a power of 86.5% at an alpha error level of 5%. A p value <0.05 was considered statistically significant.

2.2. Assessment of FMD and NMD 3. Results Ultrasound measurements were performed according to the guidelines for the ultrasound assessment of FMD of the brachial artery [12]. Using high-resolution ultrasound (General Electric (GE) Vivid 7) with a 7.5 MHz linear array transducer, diameter measurements of the right brachial artery were taken after supine rest for at least 10 min, after cuff deflation completing suprasystolic compression (50 mmHg above systolic pressure) of the right upper arm for 5 min and after sublingual application of glyceryl trinitrate (400 ␮g). A stereotactical arm was used for optimal transducer positioning on the brachial artery proximal of the bifurcation of the radial and lunar arteries. The longitudinal image of the artery was recorded at baseline, continuously from 30 s before

The two study groups were well balanced with respect to baseline features and concomitant medications (Table 1). Moreover, no significant differences were observed regarding baseline levels of BNP, IL-6, sICAM-1, sVCAM-1, FMD and NMD (Tables 1 and 2). 3.1. Flow-mediated and nitroglycerin-mediated vasodilation The results of the ultrasound measurements of the brachial artery are shown in Table 2. Vessel size and blood flow velocities were similar before and after treatment for the lev-

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Table 1 Baseline demographic, clinical and laboratory characteristics in the levosimendan and placebo groups

Men/women Ischemic/dilated Age (years) NYHA class BMI (kg/m2 ) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Left ventricular ejection fraction (%) BNP (pg/ml) IL-6 (pg/ml) sICAM-1 (pg/ml) sVCAM-1 (pg/ml) Medications Beta-blockers ACE inhibitors Aldosterone inhibitors Diuretics

Levosimendan (n = 17)

Placebo (n = 9)

p value

11/6 12/5 62 ± 10 2.7 ± 0.3 26.7 ± 2.3 102 ± 7

6/3 6/3 62 ± 11 2.6 ± 0.4 26.5 ± 1.3 104 ± 8

NS NS NS NS NS NS

67 ± 4

69 ± 5

NS

27 ± 5

28 ± 5

NS

927 ± 321 8.9 ± 4.3 339 ± 157 1386 ± 602

897 ± 295 9.9 ± 3.8 390 ± 140 1591 ± 661

NS NS NS NS NS

12 (70%) 15 (88%) 10 (58%)

6 (67%) 8 (89%) 5 (55%)

17 (100%)

9 (100%)

NYHA, New York Heart Association functional class; BMI, body mass index; BNP, B-type natriuretic peptide; IL-6, interleukin-6; sICAM-1, soluble intercellular adhesion molecule-1 and sVCAM-1, soluble vascular cell adhesion molecule-1.

osimendan and the placebo-treated group, as well as between the two groups; thus, it can be assumed that the stimulus for FMD was similar before and after each treatment and between patients of the two groups. Patients that received levosimendan had significantly increased FMD values 48 h after completion of the drug infusion as compared to the pre-treatment measurements (6.4 ± 4.4% versus 4.8 ± 3.0%; p = 0.042). In the placebo treatment group, in contrast, posttreatment FMD values were not different from baseline FMD (4.5 ± 2.1% versus 4.5 ± 2.3%; p > 0.05).

Endothelium-independent vasodilatation, assessed by NMD, was not different after treatment as compared with pre-treatment values in both levosimendan (13.7 ± 5.5% versus 13.4 ± 5.4%; p > 0.05) and placebo group (11.7 ± 5.2% versus 11.7 ± 4.8%; p > 0.05). 3.2. Markers of neurohormonal and inflammatory activation BNP, pro-inflammatory IL-6, sICAM and sVCAM levels decreased after levosimendan treatment (Table 2). On the contrary, in placebo-treated patients, none of the aforementioned neurohormonal, inflammatory and vascular-related cell adhesion molecules was altered significantly after treatment.

4. Discussion Endothelial dysfunction has recently emerged as a significant pathogenetic factor that contributes to the clinical manifestations of CHF and is associated with an unfavourable prognosis [13]. Levosimendan is a novel inodilator that is used in the short-term management of patients with severe low output CHF [14]. Experimental evidence suggests that, parallel to its principal mode of action through calcium sensitization of myofilaments, levosimendan exhibits additional unique favourable properties, such as activation of cytosplasmic membrane and mitochondrial KATP channels of a variety of cell types, including cardiac and vascular muscle cells [15–17]. On the basis of these mechanisms, levosimendan exerts a variety of systemic actions, such as vasodilatory, antiinflammatory, anti-oxidant and anti-apoptotic actions that distinguish it from the classic inotropes and possibly underlie its cardioprotective effects in patients with CHF [18]. In the present randomized placebo-controlled study, we show for the first time that levosimendan improves significantly endothelial function in patients with severe CHF, as

Table 2 Flow- and nitrate-mediated dilatation and biochemical markers before and after the therapeutic intervention Levosimendan (n = 17) Before Baseline diameter (mm) Baseline blood flow velocity (cm/s) Hyperemia diameter (mm) Hyperemia blood flow velocity (cm/s) FMD (%) NMD diameter (mm) NMD (%) BNP (pg/ml) IL-6 (pg/ml) sICAM-1 (pg/ml) sVCAM-1 (pg/ml)

4.6 57 4.9 74 4.8 5.2 13.4 927 8.9 339 1386

± ± ± ± ± ± ± ± ± ± ±

Placebo (n = 9) After

0.9 24 0.9 26 3.0 1.1 5.4 321 4.3 157 602

4.8 70 5.1 87 6.4 5.3 13.7 624 6.6 231 1134

Before ± ± ± ± ± ± ± ± ± ± ±

0.9 19 1.0 20 4.4a 1.1 5.5 262a 2.8a 75a 508a

4.4 60 4.6 87 4.5 4.5 11.7 897 9.9 390 1591

± ± ± ± ± ± ± ± ± ± ±

After 1.0 16 1.0 14 2.3 1.4 4.8 295 3.8 140 661

4.4 64 4.1 90 4.5 4.9 11.7 899 10.3 395 1657

± ± ± ± ± ± ± ± ± ± ±

1.4 20 2.0 35 2.1 1.4a 5.2 288 3.9 157 581

FMD, flow-mediated dilation; NMD, nitroglycerin-mediated dilation; BNP, B-type natriuretic peptide; IL-6, interleukin-6; sICAM-1, soluble intercellular adhesion molecule-1 and sVCAM-1, soluble vascular cell adhesion molecule-1. a Significantly different vs. the corresponding value before therapy in the same group (paired t-test or Wilcoxon’s paired test).

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indicated by the enhancement of FMD of peripheral arteries. FMD is a marker of the vasodilatory response of a vessel to an elevation of blood flow-associated shear stress [19]. Hyperemia-induced elevation of shear stress stimulates the endothelial release of a number of vasoactive substances, most predominantly NO, thereby inducing vasodilatation. Therefore, FMD is accounted as an indirect measure of endothelial NO bioavailability [19,20]. The beneficial effect of levosimendan on FMD could be attributed to the increase in blood flow, induced by the enhancement of cardiac output, which in turn leads to elevation of shear stress. The induction of NO synthesis by levosimendan is supported by a previous experimental study, according to which the hemodynamic and coronary vasodilatory effects of intracoronary administered levosimendan were abolished after simultaneous blockade of coronary NO synthase by its inhibitor l-NAME [11]. Furthermore, besides stimulating NO production, levosimendan may also lead to inhibition of NO degradation by oxygen-free radicals. Experimental results support the anti-oxidant effects of levosimendan, which inhibited H2 O2 -induced cardiac myocyte apoptosis through the activation of mitochondrial KATP channels [18]. At the clinical level, Avgeropoulou et al. showed that levosimendan reduced plasma levels of malondialdehyde, a marker of oxidative stress, in patients with advanced CHF [19–21]. In the present study, no significant alterations were observed after levosimendan infusion on endotheliumindependent NMD. However, previous experimental studies have shown that levosimendan elicits vasodilatation of peripheral and coronary arterial and venous preparations and this effect is primarily exerted through the opening of KATP channels located on cytoplasmic membranes of vascular smooth muscle cells [22–24]. These apparently conflicting results could be attributed to the different study settings (in vitro versus in vivo), as well as the dosing and route of levosimendan administration. Therefore, the effect of levosimendan on vascular smooth muscle-mediated vasodilatation in humans should be further explored. In addition, we have found that the improvement of FMD after levosimendan administration was accompanied by inhibition of vascular inflammation, as represented by the reduction in plasma levels of soluble forms of cell adhesion molecules ICAM-1 and VCAM-1. This was paralleled by the decrease in levels of IL-6, a marker of systemic inflammation, and of BNP, an index that reflects the severity of neurohormonal activation in CHF. The levosimendan-induced reduction of sICAM-1 and sVCAM-1 levels represents a significant new finding, since these molecules have been shown to play a pivotal role in the process of vascular inflammation and, also, to contribute to the pathogenesis of CHF [25]. These molecules, expressed on the surface of endothelial cells, serve as ligands for leukocytes in order to adhere to and infiltrate the vascular wall, leading to a series of cellular events such as the synthesis of inflammatory cytokines, endothelial cell apoptosis and overproduction of extracellular matrix and interstitial collagen, and ultimately cause

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pathologic vascular remodeling [26]. In CHF, plasma levels of cell adhesion molecules are elevated and correlate with the severity of the syndrome [27]. The observed levosimendan-induced attenuation of inflammatory process probably results from both a direct effect of the drug as well as from the concomitant improvement of patients’ hemodynamic condition. Previous studies have shown that levosimendan bears anti-inflammatory properties [28]. At the same time, levosimendan leads to reduction of left ventricular wall stress, while at the same time it increases cardiac output and hence peripheral tissue perfusion, which in turn leads to down-regulation of cytokine transcriptional factors, such as NF-kB. This explanation has been added to the revised version of the manuscript. Although both patients and the observers who performed ultrasound measurements and biochemical tests were not aware of the treatment status of individual patients, this study was not a completely double-blinded one. Moreover, the results of the study refer only to the short period following levosimendan administration. Further clinical trials are needed for the evaluation of the long-term effects of the drug on endothelial function and of their potential prognostic significance. In conclusion, levosimendan appears to exert beneficial effects on endothelial function in patients with advanced CHF, as shown by the improvement of endotheliumdependent vasodilatation and the down-regulation of soluble adhesion molecules. Therefore, it could offer additional therapeutic advantages in patients with advanced heart failure.

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