We studied 11 adult patients with a clinical diagnosis of plaque psoriasis who had attended our hospital's dermatology department. All patients were consecutively enrolled in the study and gave their written informed consent. Seven patients had never received systemic therapy and the other 4 had not received systemic treatment in the preceding 4 to 6 weeks. Samples were collected and tested between February and May 2012. During the same period a control group of 11 healthy volunteers matched for age and sex was selected and underwent the same tests. The exclusion criteria for patients and controls included the presence of cutaneous lymphoma or other cancers and established cardiovascular disease (heart attack or stroke).

The severity of psoriasis was assessed using the Psoriasis Area Severity Index (PASI) and the involved body surface area (BSA). The weight, height, and waist circumference of patients and controls were measured and used to calculate body mass index (BMI, kg/m2). Patients with a BMI > 25 were considered overweight. Systolic and diastolic blood pressures were measured after 10 minutes on 3 occasions, and the average value was calculated. In addition, serum levels of HDL-cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides, glucose, insulin, C-reactive protein (CRP), and fibrinogen were measured and the erythrocyte sedimentation rate (ESR) determined in both patients and controls. The criteria of the National Cholesterol Education Program's Adult Treatment Panel III (ATP-III) report were used to assess the prevalence of metabolic syndrome.25 Participants were considered to have metabolic syndrome if they fulfilled 3 or more of the following criteria: waist circumference>102cm in men and>88cm in women; ESR>10mm/h; hypertriglyceridemia>150mg/dL; HDL-C<40mg/dL in men and<50mg/dL in women; blood pressure>130/85mmHg; fasting glucose>110mg/dL.

Blood samples were collected following standard procedures (using K2-EDTA tubes and the BD Vacutainer system; BD Diagnostics) in the hospital's laboratory. Plasma was isolated by Ficoll gradient centrifugation as previously described2 and plasma aliquots were stored at -80°C until analysis.

Total RNA was isolated from 200μL of plasma from patients (n=11) and controls (n=11) supplemented with 3.5μL of 1.6 ×108 copies/μL of miRNeasy Control Serum/Plasma Spike-In (Qiagen, Catalog No. 219610), using the Qiagen miRNeasy Mini Kit (Catalog No. 21704) in accordance with the manufacturer's instructions. The quality and amount of total RNA was determined using the Experion Automated Electrophoresis System (Bio-Rad) and the NanoDrop system (Thermo Fisher), respectively. cDNA was synthesized from 1μg of total RNA using the miScript II Reverse Transcription Kit (Qiagen) following the manufacturer's recommendations. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on a CFX96 thermal cycler (Bio-Rad). Every 10μL of PCR reaction mixture contained 1X QuantiTec SYBR Green PCR Master Mix, 1X miScript Universal Primer, 1X miRNA-specific primer, and 10ng cDNA. miRNA-specific primers from Qiagen were used (Homo sapiens miR-33a, Catalog no. MS00003304; and Homo sapiens miR-126-1, Catalog No. MS00003430). The following PCR program was used: 15minutes of incubation at 95¿C (to activate the Hot Start Taq DNA polymerase) followed by 40 cycles of 15seconds at 94¿C, 30seconds at 55¿C, and 30seconds at 70¿C. The fluorescence emitted by SybrGreen was measured at the end of the extension step of each cycle. The specificity of PCR amplification was determined by performing melt curve analysis of the amplicon once the PCR was completed. This involved heating from 55¿C to 90¿C in increments of 0.5¿C every 10seconds. Quantification was performed based on the results of 3 PCR reactions per sample from each participant in the control and patient groups. The threshold cycle (Ct) was calculated using the qRT-PCR software, and was defined as the point at which the amount of amplicon detected began to increase exponentially. The results were normalized internally to the levels of miRNeasy Serum/Plasma Spike-In Control Ce-miR-39_1 (Qiagen primer, Catalog No. MS00019789). Relative transcript levels in each sample were calculated using the 2-ΔΔCT method.26

Patients (n=11) and controls (n=9) underwent color Doppler ultrasonography (Acuson Antares, Siemens) of the supra-aortic trunks using a 10-5MHz transducer to detect atheromatous plaques, as previously described.2,23 A carotid intima-media thickness (IMT)>1.5cm was considered indicative of the presence of a plaque.

Results are expressed as the median and interquartile range (IQR) unless otherwise specified. Plasma expression levels of miRNAs were analyzed using the nonparametric Wilcoxon signed rank test to compare the median of the patient group with that of the control group. Where indicated, the Mann-Whitney test was used to analyze differences between patients and controls. The Fischer test was used for the analysis of qualitative variables and the Spearman test to calculate correlations Differences were considered statistically significant at P<.05 (GraphPad Prism version 5.01, GraphPad Software Inc.).

The patient and control groups both consisted of 5 males and 6 females. While the median age was somewhat higher in the patient group, this difference was not statistically significant (Table 1). All participants were white. In the patient group the median disease activity scores (PASI and BSA) were>10 (Table 1) and the median duration of disease was 5 years.

The median IMT in patients was 0.57mm (IQR, 0.54-0.61), as compared with 0.50mm in controls (IQR, 0.48-0.54) (P=.0055). One of the patients presented carotid atherosclerotic plaques.

We observed no significant differences between patients and controls in levels of HDL-C, LDL-C, triglycerides, glucose, or insulin (Table 1). One of the patients had metabolic syndrome according to ATP-III criteria.25 Positive correlations were detected between triglyceride levels and plasma levels of glucose (r=0.7900, P=.0038, n=11), insulin (r=0.8018, P=.0030, n=11), and fibrinogen (r=0.6651, P=.0255, n=11). LDL-C levels were correlated with disease duration (r=0.8219, P=.0019, n=11), and HDL-C levels were inversely correlated with PASI score (r=-0.6391, P=.0343, n=11), BMI (r=-0.6273, P=.0388, n=11), and systolic blood pressure (r=-0.6651, P=.0255, n=11). Analysis of obesity parameters revealed no differences in BMI, ESR, or waist circumference between patients and controls (Table 1). In the patient group we observed a correlation between BMI values and waist circumference (r=0.9091, P=.0001), glucose levels, (r=0.6895, P=.0189), and insulin levels (r=0.7973, P=.0033). Levels of the inflammatory marker and coagulation protein fibrinogen were higher in patients vs controls (Table 1).

We observed a significant increase in the relative plasma expression levels of miR-33 in psoriasis patients (5.34; IQR, 3.12-7.96) with respect to controls (2.33; IQR, 1.71-2.84) (P=.0049, Wilcoxon signed rank test). Plasma levels of miR-33 in 4 of the controls were too low to be correctly quantified, and were thus quantified in only 7 of the 11 controls (Fig. 1A) A positive correlation between plasma levels of miR-33 and insulin was observed in the patient group (r=0.7289, P=.0109) but not in the control group (r = 0.1071, P = .8397, n = 7). We detected no correlation in either group between plasma miR-33 expression and clinical parameters such as weight or BMI. Insulin resistance (IR) was calculated by homeostasis model assessment (HOMA) using the following equation: HOMA-IR = [insulin×glucose]/22.5, with insulin expressed in μU/mL and glucose in mmol/L. No differences in HOMA-IR were observed between patients (1.5; IQR, 1.3-3.4) and controls (1.5; IQR, 1.2-2.02) (P = .4307, Mann-Whitney test). A positive correlation between plasma miRNA levels and HOMA-IR was observed in patients (r=0.6545, P=.0289) but not in controls (r=-0.2143, P=.6445).

Figure 1.

Plasma levels of miR-33a and miR-126 in each sample were calculated using the 2-ΔΔCT method26 (see Materials and Methods). Results are presented as the median and interquartile range in logarithmic scale (base 10). Anti-logarithmic values are indicated in the vertical axes. The Wilcoxon signed rank test was used to compare the median of the patient group (n=11) with that of the control group (n=11). Plasma levels of miR-33 expression were lower in controls than in psoriasis patients (a). Plasma miR-33 levels were quantified in 7 of the 11 controls studied (see Results). Analyses were performed using Graph-Pad Prism software (version 5.01).

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We observed no significant increase in the relative expression levels of miR-126 in the plasma of patients (0.5384; IQR, 0.2839-1.052) vs controls (0.361; IQR, 0.2806-0.5371]) (P=.0830).

A negative correlation between levels of circulating miR-126 and IMT values was observed in patients (r =-0.6181, P=.0426) but not in controls (r=-0.3333, P=.3807).

Finally, in the group of psoriasis patients we observed no significant correlations between plasma expression levels of miR-33 or miR-126 and the other clinical parameters analyzed.

The authors declare that no experiments were performed on humans or animals during the course of this study.

The authors declare that they have followed the protocols of their place of work concerning the publication of patient data and that all patients included in this study were appropriately informed and gave their written informed consent.

The authors declare that no private patient data appear in this article.