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. Author manuscript; available in PMC: 2015 Mar 1.
Published in final edited form as: J Invest Dermatol. 2013 Aug 28;134(3):635–642. doi: 10.1038/jid.2013.359

Anti-psoriatic therapy recovers high-density lipoprotein composition and function

Michael Holzer 1, Peter Wolf 2, Martin Inzinger 2, Markus Trieb 1, Sanja Curcic 1, Lisa Pasterk 1, Wolfgang Weger 2, Akos Heinemann 1, Gunther Marsche 1
PMCID: PMC4178282  EMSID: EMS60335  PMID: 23985995

Abstract

Psoriasis is a chronic inflammatory disorder associated with increased cardiovascular mortality. Psoriasis affects high-density lipoprotein (HDL) composition, generating dysfunctional HDL particles. However, data regarding the impact of anti-psoriatic therapy on HDL composition and function are not available.

HDL was isolated from 15 psoriatic patients at baseline and after effective topical and/or systemic anti-psoriatic therapy and from 15 age- and sex-matched healthy controls. HDL from psoriatic patients showed a significantly impaired capability to mobilize cholesterol from macrophages (6.4 vs. 8.0% [3H]-cholesterol efflux, p<0.001), low paraoxonase (350 vs. 217 μM/min/mg protein, p=0.011) and increased Lp-PLA2 activities (19.9 vs. 12.1 nM/min/mg protein, p=0.028). Of particular interest, the anti-psoriatic therapy significantly improved (i) serum lecithin-cholesterol acyltransferase activity and decreased total serum lipolytic activity but did not affect serum levels of HDL-cholesterol. Most importantly, these changes were associated with a significantly improved HDL-cholesterol efflux capability.

Our results provide evidence that effective anti-psoriatic therapy recovers HDL composition and function, independent of serum HDL-cholesterol levels and support to the emerging concept that HDL function may be a better marker of cardiovascular risk than HDL-cholesterol levels.

Introduction

Epidemiological and clinical studies have consistently shown that psoriasis is associated with an increased cardiovascular risk (Armstrong et al, 2013, Mehta et al, 2010). Psoriasis, a widespread chronic inflammatory disease, affects about 2 – 3% of the population. Although generally characterized by typical lesions on the skin of the trunk, extremities and scalp, psoriasis also affects the entire organism by maintaining a low-grade inflammatory status. Traditional risk factors for cardiovascular disease, such as hypertension, elevated C-reactive protein and obesity, are more frequent in psoriatic patients than in the normal population (Neimann et al, 2006, Kaplan, 2008). Patients with psoriasis are more likely to have a deteriorated lipid profile, with higher triglyceride levels and significantly decreased HDL-cholesterol (Rocha-Pereira et al, 2001).

It is thought that HDL protects against cardiovascular disease by removing cholesterol from artery wall macrophages in a process called reverse cholesterol transport. In addition, HDL exerts additional anti-atherogenic effects, such as anti-oxidative activities (Kontush and Chapman, 2010). Despite the clear epidemiological evidence that plasma levels of HDL-cholesterol are inverse and independent predictors of cardiovascular disease risk, genetic studies have yielded inconsistent data (Voight et al, 2012). Moreover, raising HDL-cholesterol by the cholesteryl-ester transfer protein (CETP) inhibitors torcetrapib and dalcetrapib did not translate into cardiovascular protection (Landmesser et al, 2012) supporting to the emerging concept that HDL function is a better marker than HDL-cholesterol levels. In line with this assumption, a recent study clearly showed that HDL cholesterol efflux capacity, independently of HDL cholesterol levels, was inversely associated with the risk of coronary artery disease (Khera et al, 2011). Given that inflammation alters HDL particles in terms of structure, size, composition and metabolism, it is becoming increasingly apparent that direct measures of HDL function are needed rather than relying on surrogate markers such as the concentration of HDL-cholesterol (Shah et al, 2013, Triolo et al, 2013, Marsche et al, 2013). Recent work from our group has shown that psoriasis alters HDL composition and function (Holzer et al, 2012), reflecting a shift to a pro-atherogenic profile, associated with an impaired cholesterol efflux capacity of HDL.

In the present study, we investigated whether anti-psoriatic therapy affects HDL function. Our study included paired measurements of patients with disease and multiple measures of HDL function and composition. For that purpose, we isolated HDL of healthy subjects and psoriasis patients before and after anti-psoriatic therapy and assessed HDL functionality.

Results

Anti-psoriatic therapy does not alter blood lipid levels

HDL was isolated from 15 psoriasis patients at baseline and after anti-psoriatic therapy and from 15 age- and sex-matched controls. Clinical characteristics, medical history and individual treatments plans are given in Table 1, Supplemental Table 1 and Supplemental Table 2. Evaluation of the psoriasis area and severity index (PASI) clearly indicated a significant improvement in diseases severity over the treatment period (Table 1, Figure 1a, Supplemental Table 2), without affecting body weight of patients. Circulating C-reactive protein levels in the treatment group tended to decrease, but did not reach statistical significance (Table 1). Anti-psoriatic therapy did not alter blood lipid levels in the treatment group and HDL-cholesterol levels remained significantly lower than compared to the control group (Table 1).

Table 1. Clinical characteristics of study subjects.

Controls Psoriasis
baseline after anti-psoriatic treatment
n 15 15 15
Age (yr) 43 (35.0-60.1) 49.7 (42.1-65.4) 50.7 (43.3-67.3)
Male/Female 10/5 12/3 12/3
PASI - 16.6 (10.7-21.3) 6 (2.4-11.8)†
CRP (nmol/L) 2.9 (0-3) 59 (5-125) 41 (4-101)
Total cholesterol (mmol/L) 5.2 (4.4-5.9) 5.3 (4.1-5.7) 4.8 (4.0-5.5)
Triglycerides (mmol/L) 1.3 (0.9-2.2) 1.8 (0.8-2.1) 1.7 (0.8-2.8)
HDL-cholesterol (mmol/L) 1.7 (1.3-2.0) 1.2 (0.9-1.4)* 1.1 (0.9-1.2)*
LDL-cholesterol (mmol/L) 3.0 (2.3-3.5) 3.1 (2.1-4.1) 3.0 (2.1-3.8)
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Results are given as medians with interquartile range. PASI, psoriasis area and severity index CRP, C-reactive protein.

*

p < 0.05 vs. control;

†

p < 0.05 vs. baseline.

Figure 1. Psoriasis impairs cholesterol efflux capability of HDL.

Figure 1

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(a) Psoriasis area and severity index (PASI) score at baseline and after anti-psoriatic therapy. (b) HDL (50 μg/ml protein) from 15 control subjects and 15 psoriatic patients at baseline and after anti-psoriatic therapy were examined for its ability to induce [3H]cholesterol efflux from RAW264.7 macrophages. Cholesterol efflux is expressed as radioactivity in the supernatant relative to total radioactivity. Values shown represent means of two individual experiments performed in duplicate. (c) ApoB-depleted serum (2.8%) was examined for its ability to induce [3H]cholesterol efflux from cAMP-stimulated RAW264.7 macrophages. Values shown represent means of two individual experiments performed in duplicate. The Spearman correlation coefficient is noted. (d) HDL-mediated [3H]cholesterol efflux from RAW264.7 macrophages at baseline and after anti-psoriatic therapy. (e) Correlation between PASI and [3H]cholesterol efflux induced by HDL. (f) Correlation between PASI and apoB-mediated [3H]cholesterol efflux. The Spearman correlation coefficients are noted with its significance level.

Effect of anti-psoriatic therapy on HDL-mediated cholesterol efflux

To compare the functionality of HDL particles before and after anti-psoriatic therapy, we measured HDL-mediated cholesterol efflux for each subject using RAW264.7 macrophages. In line with our previous study (Holzer et al, 2012), we observed a reduction of cholesterol efflux capacity of HDL from psoriatic patients when compared with control subjects. Most importantly, cholesterol efflux capability of HDL improved significantly after anti-psoriatic therapy (Figure 1b, d) and correlated negatively with the PASI score (Figure 1e). When baseline and therapy measurements were analysed separately, we observed that at baseline a borderline correlation between cholesterol efflux and PASI was present (r=-0.483, p=0.068), whereas after treatment the correlation was significant (r=-0.621, p=0.013).

To validate results obtained with mouse RAW264.7 macrophages, cholesterol efflux experiments were performed using human lipid-laden THP1 macrophages. Experiments in human THP1 macrophages revealed comparable results (Supplemental Figure 1a, b) and a significant correlation between efflux measures in RAW264.7 and THP1 macrophages was present (Supplemental Figure 1c).

Anti-psoriatic therapy may affect different HDL subpopulations. During the ultracentrifugation procedure to isolate mature HDL, a part of lipid-poor pre-beta-1 particles strips off which are preferred acceptors for ABCA1-mediated cholesterol efflux (de la Llera-Moya et al, 2010). To gain further insights whether anti-psoriatic therapy also affects pre-beta-1 particles, cholesterol efflux capability of apoB-depleted serum (containing all HDL subpopulations) was assessed using ABCA1 upregulated RAW264.7 macrophages. In this particular assay, pre-beta-1 HDL is the main driver of ABCA1-mediated cholesterol efflux even in the presence of all other HDL subpopulations (Asztalos et al, 2005). In good agreement with a recent study (Holzer et al, 2012, Mehta et al, 2012), we observed that cholesterol efflux capacity of apoB-depleted serum was significantly lower in psoriatic patients (Figure 1c). However, in contrast to efflux experiments performed with isolated HDL, we did not observe an improvement of cholesterol efflux capability of sera after anti-psoriatic therapy (Figure 1c), and cholesterol efflux capability of apoB-depleted sera did not correlate with PASI (Figure 1f).

Anti-psoriatic therapy alters HDL composition

We next analyzed the phospholipid content of isolated HDL, given that the phospholipid content is critical for cholesterol efflux capacity of mature HDL (Sankaranarayanan et al, 2009, Holzer et al, 2011a). Of note, anti-psoriatic therapy tended to increase HDL-phospholipid content to levels observed in HDL of the control group (Figure 2a). The HDL-phospholipid content significantly correlated with cholesterol efflux capacities of HDL within the control and the psoriasis groups (Figure 2b).

Figure 2. HDL-phospholipid correlates with the cholesterol efflux capability of HDL.

Figure 2

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(a) Quantification of phospholipid content in isolated HDL from healthy controls and psoriasis patients at baseline and after anti-psoriatic therapy. Results represent duplicate measurements of three independent experiments. (b) Correlation between HDL-mediated [3H]-cholesterol efflux from macrophages and the HDL-phospholipid content (HDL-PL) Spearman correlation coefficients are noted for each plot.

Anti-psoriatic therapy increases lecithin-cholesterol acyltransferase (LCAT) activity and reduces total serum lipolytic activity

Prompted by the observation that the anti-psoriatic therapy is associated with compositional alterations of HDL, we assessed whether anti-psoriatic therapy affects phospholipid transfer protein (PLTP), LCAT, CETP and total lipase activities of sera to assess the underlying mechanism(s). Interestingly, we observed that anti-psoriatic therapy did not alter serum PLTP (Figure 3a) and CETP (Figure 3b) activities, whereas LCAT activity was significantly increased (Figure 3c). In contrast to LCAT activity, the total serum lipolytic activity in patients after anti-psoriatic therapy was significantly decreased (Figure 3d). In agreement with increased LCAT activity and decreased serum lipolytic activity, an increased HDL subpopulation with large particle size (12-16nm) was observed by native gel electrophoresis (Figure 3e).

Figure 3. Anti-psoriatic therapy reduces serum lipase activity.

Figure 3

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Quantification of (a) phospholipid transfer protein (PLTP) and (b) cholesteryl-ester transfer protein (CETP) (c) lipase and (d) lecithin-cholesterol acyltransferase (LCAT) activity. Enzymatic activities where measured in serum of psoriasis patients at baseline and after anti-psoriatic therapy with commercial available kits as described in the method section. Results represent measurements of three independent experiments. (e) Pooled HDL samples were separated by native gel electrophoresis and stained with Coomassie Brillant Blue G-250. A gel image was analyzed with ImageJ software to obtain an intensity plot. Standard proteins are indicated as dotted lines with their size in nm.

Effects of anti-psoriatic therapy on paraoxonase and lipoprotein-associated phospholipase A2 activities

Paraoxonase, an important HDL-associated enzyme, has been implicated in anti-oxidant and anti-inflammatory functions (Mackness and Mackness, 2010, Aviram, 2011) and its activity has been related to cardiovascular risk (Bhattacharyya et al, 2008). We observed that paraoxonase activity of HDL from psoriasis patients was significantly lower, when compared to controls. Another HDL-associated enzyme, lipoprotein-associated phospholipase A2 (Lp-PLA2), has recently been associated with an enhanced risk of coronary artery disease (Rosenson and Stafforini, 2012). Lp-PLA2 activity is significantly increased in psoriasis patients and patients suffering from end-stage renal disease (Holzer et al, 2012, Holzer et al, 2011a). In line with our previous study (Holzer et al, 2012), we observed that the activity of Lp-PLA2 was significantly increased in the psoriasis group. Interestingly, anti-psoriatic therapy tended to recover HDL-paraoxonase and Lp-PLA2 activities (Figure 4a, 4b).

Figure 4. Anti-psoriatic therapy increases paraoxonase activity and reduced Lp-PLA2 activity on isolated HDL.

Figure 4

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(a, b) Activity of HDL-associated paraoxonase was measured using paraoxon as substrate. (c, d) Lipoprotein associated phospholipase A2 (Lp-PLA2) activity of HDL was measured using 2-thio PAF as substrate. Paraoxonase and Lp-PLA2 activities of HDL were calculated from the slopes of the kinetic chart of three independent experiments. (e, f) The anti-oxidative activity of HDL was determined by inhibition of AAPH-initiated oxidation of the fluorescent dye dihydrorhodamine (DHR). Incubation of DHR in presence of HDL from healthy subjects (control) or psoriasis patients (psoriasis) led to a reduction in the oxidation of DHR. Results represent measurements of three independent experiments.

HDL exhibits unique anti-oxidative activities (Kontush and Chapman, 2010). To assess the anti-oxidative activity of HDL, we monitored the ability of HDL to inhibit oxidation of the fluorescent dye dihydrorhodamine. We observed that HDL from controls and psoriasis patients is similarly potent in inhibiting radical-induced oxidation of dihydrorhodamine, suggesting that anti-oxidant activity of psoriatic HDL is not significantly altered (Figure 4c).

Discussion

Several clinical studies have shown that psoriatic patients face an increased burden of cardiovascular disease (Armstrong et al, 2013, Mehta et al, 2010), which cannot fully be explained by traditional risk factors (Neimann et al, 2006). Recent work from our group and others has shown that psoriasis alters HDL composition and function (Holzer et al, 2012), reflecting a shift to a pro-atherogenic profile, associated with an impaired cholesterol efflux capacity of isolated HDL.

The data presented here describe the novel observation that an effective anti-psoriatic therapy, as shown by a significant improvement in PASI score, markedly recovers HDL function. We performed cholesterol efflux experiments with isolated HDL in two macrophage cell lines (RAW264.7, THP1) and observed that anti-psoriatic therapy significantly improved HDL cholesterol efflux capacity. Notably, anti-psoriatic therapy showed no effect on HDL or LDL-cholesterol levels, providing evidence that HDL function is not adequately reflected by HDL-cholesterol levels. Efflux of cholesterol is regulated by the phospholipid content and size of larger, spherical HDL particles that are the preferred acceptors of the cholesterol released from cells by ABCG1 and scavenger receptor BI (Sankaranarayanan et al, 2009, Yancey et al, 2000). Interestingly, anti-psoriatic therapy significantly increased the phospholipid content of HDL that correlated with the cholesterol efflux capability, providing support to the concept that the phospholipid content is critical for cholesterol efflux capacity of mature HDL (Sankaranarayanan et al, 2009, Holzer et al, 2011a). Of particular interest, we observed that anti-psoriatic therapy was associated with increased LCAT activity and reduced serum lipolytic activity, whereas CETP and PLTP activities were not altered. These data suggest that activities of LCAT and serum lipases are modulated by low-grade inflammation and are recovered by anti-psoriatic therapy. Of particular interest, these anti-psoriatic therapy associated alterations in serum enzymatic activities were accompanied by an increased size of larger, spherical HDL particles particle size.

A recent study has provided compelling evidence that cholesterol efflux of ABCA1-upregulated macrophages promoted by apoB-depleted serum is inversely associated with the risk of coronary artery disease (Khera et al, 2011). In line with a recent report (Mehta et al, 2012), we observed that cholesterol efflux capacity of apoB-depleted serum is reduced in psoriasis patients using the same cholesterol efflux assay. Interestingly, anti-psoriatic therapy did not improve cholesterol efflux capability of apoB-depleted serum. However, it should be noted that cholesterol efflux to apoB-depleted serum during the reported cholesterol efflux assay mainly reflects the cholesterol acceptor activity of the lipid-poor apoA-I pool via ABCA1. Mature HDL-particles serve as only a minor acceptor (≈40%) of radiolabeled cholesterol within apoB-depleted serum (Li et al, 2013). Enhanced LCAT activity is associated with the formation of large HDL particles, whereas LCAT deficiency leads to accumulation of nascent HDL due to impaired maturation of HDL particles (Kunnen and Van Eck, 2012). Therefore, it might be assumed that anti-psoriatic therapy associated increases in LCAT activity on the one hand forms large HDL particles with increased cholesterol acceptor capability but on the other hand reduces lipid-poor pre-beta-1 HDL levels, resulting in unaltered cholesterol acceptor capability of apoB depleted serum. In agreement with that assumption is the fact that sera of carriers of LCAT gene mutations have unaltered capacities to promote cholesterol efflux. Efflux driven by low levels of large, spherical HDL is compensated by increased efflux driven lipid-poor pre-beta-1 HDL (Kunnen and Van Eck, 2012). Therefore it has to be noted that our finding of a recovery of HDL cholesterol-efflux capacity by anti-psoriatic therapy reflects mainly alterations in the composition and function of mature HDL.

Other proteins such as paraoxonase that cotransport with HDL in serum are well-known to have anti-atherogenic properties. In contrast to our previous study (Holzer et al, 2012), we observed in the present study that paraoxonase activity in psoriasis patients was significantly lower when compared to healthy controls. This apparently contrasting finding may be explained by a higher mean PASI score of the psoriatic patients of our present study (17.6 vs. 11.4), suggesting that a higher inflammatory state is linked to lower paraoxonase activity, as suggested by others (Toker et al, 2009). In line with our previous study (Holzer et al, 2012), we observed that the activity of HDL-associated Lp-PLA2 was significantly increased in the psoriasis group. Interestingly, anti-psoriatic therapy tended to recover HDL-paraoxonase and Lp-PLA2 activities, suggesting that other potential anti-atherogenic properties of HDL are recovered, at least in part. Interestingly, we observed that control HDL and HDL from psoriatic patients at baseline and after therapy inhibited DHR oxidation to a similar extent although paraoxonase activity was significantly altered. Given that Lp-PLA2 activity was significantly increased in psoriatic HDL (and LCAT activity after anti-psoriatic therapy), low paraoxonase activity of psoriatic HDL might be compensated by increased Lp-PLA2 and LCAT activities with similar anti-oxidant activity. Interestingly, a recent study provided evidence that paraoxonase prevents LCAT inactivation (Hine et al, 2012), hence improvement of paraoxonase activity through anti-psoriatic therapy might trigger, at least in part, increased LCAT activity. However, much more research into the HDL antioxidant system needs to be done to dissect the exact mechanism(s) at work.

To provide indications about the efficacy of different anti-psoriatic therapies on PASI and HDL parameters, we provide data of individual patients and the effectiveness of their therapy shown in the Supplemental Table 3. This data suggests that all therapies are effective in reducing PASI, whereby differences in HDL parameters seemed to be most pronounced in patients receiving systemic therapies (TNF inhibition, anti-IL-23 and other systemic) (Supplemental Table 3).

However, several study limitations have to be noted. The lack of a control arm consisting of patients with psoriasis that do not receive anti-psoriatic therapy over the same time period does not allow proving a cause-and-effect relationship within this explorative study. Our study is limited by the correlative nature, not permitting causal inference. Moreover, due to the small sample size, we cannot exclude the possibility that potential significant differences are missed. Therefore, further studies in larger cohorts are warranted to confirm our results.

In summary, our results provide novel evidence that effective anti-psoriatic therapy is able to improve HDL function. Of particular interest in this regard is the recent observation that anti-inflammatory therapies of psoriasis patients improve cardiovascular outcome (Ahlehoff et al, 2013). Hence, anti-psoriatic therapy associated improvements of HDL function, as observed in the present study, might have contributed to the cardiovascular protection and lend support to the emerging concept that HDL function, such as cholesterol efflux capacity, may be a better marker of cardiovascular risk than HDL cholesterol concentration.

Materials and Methods

Characteristics of study subjects and blood collection

Blood was sampled from patients with moderate to severe chronic plaque-type psoriasis and healthy volunteers after obtaining written informed consent, according to a protocol approved by the Institutional Review Board of the Medical University of Graz (Nr.: 21-523 ex 09/10). The clinical characteristics and medical history of study subjects are given in Table 1 and Supplemental Table 1. The current investigation is based on the re-analysis of the pre-treatment serum available from ten patients of a previous report (Holzer et al, 2012) and five newly enrolled patients and newly sampled blood after anti-psoriatic treatment in all patients. At the time of the after-treatment investigation, the patients continued to be on or had completed treatment. The primary treatment modalities included UVB-311nm phototherapy (n=3), oral psoralen plus UVA (PUVA) photochemotherapy (n=1), adalimumab (n=1), etanercept (n=2), ustekinumab (n=2), methotrexat (n=2), cyclosporin and fumaric acid (n=1), S1P1 agonist (n=1), and topical dithranol (n=2). Additional topical treatment with calcipotriol (n=7), tacalcitol (n=1), dithranol (n=3), and/or steroids (n=11) was given in 12 of the 15 patients. A list with concise information about treatment of individuals can be found in Supplemental Table 2. The time from baseline to after anti-psoriatic treatment investigation of lipoprotein levels and functions was 76 weeks (SD ± 14.7).

Isolation of HDL

Serum density was adjusted with potassium bromide (Sigma, Vienna, Austria) to 1.24 g/ml and a two-step density gradient was generated in centrifuge tubes (16 × 76 mm, Beckman) by layering the density-adjusted plasma (1.24 g/ml) underneath a KBr-PBS-density solution (1.063 g/ml) (Holzer et al, 2012). Tubes were sealed and centrifuged at 90.000 rpm for 4 hours in a 90Ti fixed angle rotor (Beckman Instruments, Krefeld, Germany). After centrifugation, the HDL-containing band was collected, desalted via PD10 columns (GE Healthcare, Vienna, Austria) and immediately used for experiments or stored at −70°C.

Determination of plasma and HDL lipid composition

Levels of total cholesterol and phospholipids (Diasys, Holzheim, Germany) were measured enzymatically. LDL cholesterol was calculated according to the Friedewald equation using HDL cholesterol values measured in the supernatant of the phosphotungstic acid/MgCl2 precipitation.

Cholesterol efflux capability of HDL

RAW264.7 macrophages (7×106 cells/well), maintained in DMEM with 10 % fetal bovine serum (FBS), were plated in 48-well plates and grown overnight. Cells were labeled for 24 hours with [3H]cholesterol (1 μCi/mL) in medium containing 5% FBS, 50 μg/ml aggregated LDL protein and the LXR agonist TO-901317 (2 μmol/L). After labeling, cells were rinsed and equilibrated in serum-free media containing 0.2 % bovine serum albumin for 2 hours. To determine [3H]cholesterol efflux, cells were incubated with 50 μg/ml HDL protein for 3 hours at 37°C. Afterwards, the supernatant was collected and used for liquid scintillation counting.

THP-1 macrophages were maintained in RPMI 1640 with 10 % fetal bovine serum (FBS). Cholesterol efflux experiments were performed as described above.

Cholesterol efflux capability of apoB-depleted serum

ApoB-depleted serum was prepared by addition of 40μl polyethylenglycol (20% in 200 mmol/L Glycine buffer) to 100 μl serum with gentle mixing. Serum was incubated at room temperature for 20 minutes and the supernatant recovered after centrifugation (10,000 rpm, 20 minutes, 4°C). RAW 264.7 macrophages were plated with DMEM containing 10% FBS in 48-well plates, cultured for 24 hours and loaded with 1 μCi/mL radiolabeled [3H]cholesterol (Perkin Elmer, Boston, MA, USA) in serum containing media for 24 hours. The day after labeling, the cells were washed and stimulated with 0.3 mmol/L 8-(4-chlorophenylthio)-cyclic AMP (Sigma, Darmstadt, Germany) in serum-free media for 6 hours to upregulate ABCA1 as described (Holzer et al, 2011a, Holzer et al, 2011b). Subsequently, cells were washed and efflux measured towards 2.8% apoB–depleted serum in serum-free media for 4 hours. All steps were performed in the presence 2 μg/mL of the acyl coenzyme A:cholesterol acyltransferase inhibitor Sandoz 58-035 (Sigma, Darmstadt, Germany).

Paraoxonase activity assay

Ca2+-dependent paraoxonase activity was determined with a photometric assay using phenylacetate as the substrate as described (Holzer et al, 2012). HDL (5 μg protein) was added to 200 μL buffer containing 100 mmol/L Tris, 2 mmol/L CaCl2 (pH 8.0) and paraoxon (1 mmol/L). The rate of hydrolysis of paraoxon was monitored by the increase of absorbance at 405 nm and readings were taken every 150 seconds at room temperature to generate a kinetic plot. The slope from the kinetic chart was used to determine ΔAb270nm / min. Enzymatic activity was calculated with the Beer-Lambert Law from the molar extinction coefficient of 17100 L*mol−1*cm−1 PON activities were expressed as mmol 4-nitrophenol formed per minute per mg HDL protein.

Lp-PLA2 activity assay

Lp-PLA2 activity was measured with a commercially available photometric assay (Cayman Europe, Talinn, Estonia) using 2-thio PAF as substrate as described (Holzer et al, 2012).

PLTP, CETP, LCAT and total serum lipase activity assays

Commercial available kits were used to assess PLTP (Eubio, Vienna, Austria), CETP (Eubio, Vienna, Austria), LCAT (Merck, Darmstadt, Germany) and total lipase activity (Cayman Europe, Tallinn, Estonia) in sera according to the manufactures instructions. Since LCAT activity is measured as the ratio between wavelengths (470nm/390nm), we present activity values as relative index for easier interpretation, where 1 represents the mean activity of psoriasis patients at baseline,

Determination of the anti-oxidative capacity of HDL

The anti-oxidative activity of HDL was determined as previously described with modifications (Holzer et al, 2012). Briefly, 7.5 μg HDL protein was placed in a 384-well, 15μl of 50μmol/L DHR reagent containing 1 mmol/L 2,2′-azobis-2-methyl-propanimidamide-dihydrochloride was added. The increase in fluorescence (538nm) per minute was determined for samples containing only DHR and for samples containing DHR and individual HDL samples from healthy controls or psoriasis patients.

HDL particle size analysis

Pooled fraction of HDL (5 μg protein per lane) from psoriasis patients at baseline and after therapy were separated by gradient gel electrophoresis (4–16% NativePage; Life Technologies, Vienna, Austria) under nonreducing and nondenaturing conditions.

Gels were run for 120 min at constant voltage of 150 V, in NativePage running buffer (Life Technologies, Vienna, Austria). Afterwards, gels were fixed with 25% isopropanol/10% acetic acid for 10min and stained overnight with Coomassie Brilliant Blue G-250 (Thermo Scientific, Rockford, USA). To determine the size distribution of isolated HDL, an image of the gel was analyzed with ImageJ software. Intensity blots of individual samples were obtained and compared to a high molecular weight marker (NativeMark, Life Technologies, Vienna, Austria), containing bovine serum albumin (7.1 nm), lactate dehydrogenase (8.2 nm), B-phycoerythrin (10.5 nm, apoferritin band 1 (12.2 nm) and apoferritin band 2 (18.0 nm), to estimate the size of HDL.

Statistical analysis

Statistical analyses were performed with non-parametric tests due to the small sample size. Differences in plasma and HDL parameters between control subjects and psoriasis patients were analyzed with the Kruskal-Wallis test, while differences between psoriasis patients before and after therapy were tested with the Wilcoxon signed rank test for paired observations. All correlations between compositional and functional data were determined with the use of Spearman product–moment estimates. Significance was accepted at *P<0.05, **P<0.01 and ***P<0.01. Statistical analyses were performed with PASW Statistics Version 19.

Our study (n = 15 vs n = 15) provided greater than 90% power to detect a 10% difference in cholesterol efflux capability of HDL based on our hypothesis that we would observe differences similar to those between healthy subjects and psoriasis patients as described in our previous study (Holzer et al, 2012).

Supplementary Material

Supplement

Acknowledgement

The authors thank Sabine Kern and Isabella Bambach for technical support.

Sources of Funding This work was supported by the Austrian Science Fund FWF (Grants P21004-B02, and P22976-B18 to G.M., P-22521-B18 to A.H., and W1241 to G.M., P.W. and A.H.) and by the Oesterreichische Nationalbank, Jubiläumsfond (Grant No. 14853).

Abbreviations

HDL

high-density lipoprotein

LDL

low-density lipoprotein

Lp-PLA2

lipoprotein-associated phospholipase A2

PLTP

phospholipid transfer protein

CETP

cholesteryl-ester transfer protein

PASI

psoriasis area and severity index

Footnotes

Conflict of interest None.

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