Statin therapy upregulates arachidonic acid status via enhanced endogenous synthesis in patients with plaque psoriasis

Abstract

Circulating fatty acids (FA) may be important in the psoriatic pro-inflammatory phenotype. FADS1 converts linoleic acid (LA) to arachidonic acid (AA), a precursor to potent signaling molecules. HMG-CoA reductase inhibitors (statins) increase FADS1/2 expression in vitro. Psoriasis patients (42 ± 14 years/age, 47% male) were randomized to 40 mg of atorvastatin (n = 20) or nothing (n = 10) for two weeks and plasma FA measured pre and post treatment. After treatment, LDL-C was 44% lower in the statin compared to the no-treatment group. Statins increased FADS1/2 expression, and lowered LA 12% (33% – > 29%, p<0.001) and raised AA 14% (7.7% – > 9.0%, p<0.01) with no change in the no-treatment group. In psoriasis, statins enhance AA and decrease LA, consistent with the action of enhanced FADS expression in vivo. Therapies intended to blunt the effects of AA on platelet aggregation, such as aspirin or omega-3 fatty acids, may require dose adjustment when co-administered with atorvastatin.

NCT: NCT03228017

1. Introduction

Psoriasis is a chronic inflammatory condition of the skin which is shown to increase traditional cardiovascular (CV) risk factors such as dyslipidemia and adiposity in addition to atherosclerotic cardiovascular disease (ASCVD) [1]. The underlying pathophysiology of psoriatic lesions includes immune cell activation (including T-cells) promoting cytokine production and driving keratinocyte hyper proliferation [1]. In addition to immune cell infiltrates, lesional psoriatic skin has an overabundance of lipid and fatty acid mediators including both arachidonic and linoleic acid derived metabolites [2]. Some psoriasis studies also suggest a relationship between fatty acid composition and psoriasis severity and conversely, lower serum levels of both arachidonic and linoleic acid compared to controls, highlighting the importance of fatty acid composition and a potential role of bioactive fatty acids in psoriasis [3].

HMG-CoA reductase inhibitors (statins) dramatically reduce circulating low-density lipoprotein cholesterol (LDL-C) and clinical ASCVD via a reduction in low-density lipoprotein cholesterol (LDL-C) synthesis and compensatory up-regulation in hepatic LDL-C receptors [4]. In non-inflammatory populations and in vitro experiments, statins increase long-chain polyunsaturated fatty acids with enhanced arachidonic acid (AA) synthesis from endogenous linoleic acid (LA), possibly contributing to the “pleotropic” anti-inflammatory impact of statins [5]. In psoriasis, given the systemic inflammation, pattern of dyslipidemia, and lipidomic signature within psoriatic lesions, statin therapy may improve psoriatic lesional skin severity in addition to their known impact in ASCVD reduction [6]. Therefore, as part of a randomized controlled trial (NCT03228017) to investigate CV risk in psoriasis, we evaluated the impact of 2 weeks of 40 mg of atorvastatin therapy on serum fatty acid composition in patients with psoriasis.

2. Patients and Methods

2.1. Study population

Patients with a diagnosis of psoriasis without clinical ASCVD and age-, and gender-matched controls were recruited as part of an ongoing study (NCT03228017) investigating vascular endothelial health in psoriasis from New York University (NYU) Langone Health dermatology, phototherapy, and psoriatic arthritis specialty clinics between September 2017 and April 2019 [7–9]. The methodology and primary study endpoints have previously been reported. Inclusion criteria included ≥1% body surface area of psoriasis or psoriatic arthritis with ≥1 swollen/tender joints while exclusion criteria included those with a recent (<1 month before study enrollment) change or planned change in psoriasis therapy. The study protocol was approved by the NYU School of Medicine Institutional Review Board (i17–00,692) (Supplement Fig. 1). All subjects provided written informed consent before participation in-line with the Declaration of Helsinki.

2.2. General study protocol

After overnight fasting, psoriasis participants (and controls) underwent a medical history, blood pressure, heart rate, anthropometrics, and peripheral blood collection all using established protocols [10,11]. Lipid profiles were performed at the NYU clinical laboratory (Abbott Architect System). Psoriasis participants who agreed to the interventional study component were then randomized using a random number generator to 40 mg of atorvastatin/day or no-treatment and a repeat assessment occurred at week two, as part of an ongoing study to assess aspirin and statin use in psoriasis patients: the results and protocol of which, have already been published. [7,9]

Fatty acid percent composition was performed on frozen plasma. Analytical procedures followed best practices and were as described elsewhere [12]. Briefly, fatty acids were extracted and converted to fatty acid methyl esters for analysis. High resolution gas chromatography-flame ionization detection was used for quantitative analysis with calibration by an equal weight external standard. Data are expressed as% by weight of total fatty acids and are reflected as the fatty acid composition as a percent of the whole.

2.3. RNA transcriptomic sequencing

Peripheral blood samples were collected in PAXgene Blood RNA tubes (PreAnalytiX, Qiagen/BD) with automated RNA extraction using a QIAsymphony PAXgene Blood RNA Kit (PreAnalytiX, Qiagen/BD). Prior to RNA sequencing, yield, quantity and quality of the RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). RNA sequencing libraries were generated with the Illumina TruSeq (Sand Diego, CA) and 200 ng total RNA used as starting input per sample. Samples underwent 12 cycles of amplification. Completed libraries were quantitated, normalized, and pooled. Pooled libraries were run on 2 lanes of single read 50 on the Illumina Hiseq 4000 sequencer.

Sequencing reads were mapped to the human reference genome (GRCh37/hg19) using the STAR aligner (v2.5.0c). Read count tables were generated using HTSeq (v0.6.0), normalized based on their library size factors using DESeq (v3.7), and paired differential expression analysis was performed. Pathway and gene set enrichment analysis was performed using ClusterProfiler R package (v3.6.0). All downstream statistical analyses performed in R environment (v3.1.1) (http://www.r-project.org/). Results are reported in normalized count values (NCV) with methodology that has been previously reported. [7,8,13].

2.4. Statistical analysis

Data are reported as mean ± SEM or SD where appropriate. Non- normally distributed data are reported as median and IQR (Q1, Q3). Statistical significance was determined between study groups using parametric and non-parametric tests as appropriate. Paired-sample t- or Wilcoxon tests for changes between baseline and follow-up data points were also performed as appropriate. Statistical significance was determined using a two-tailed alpha <0.05 with all analyses performed in Stata v. 14 (College Station, TX: StataCorp LP).

3. Results

Fifty-six participants were evaluated: 40 with psoriasis and 16 matched controls. Demographics and clinical characteristics are presented in supplement Table 1 in those with psoriasis and controls. Age, sex, race/ethnicity, body mass index, blood pressure and CV risk were similar between groups. On average the psoriasis patients had moderate psoriasis severity with about 1/3rd on biologic therapy and 18 years psoriasis disease duration. There was no difference in baseline serum fatty acid composition between psoriasis and control subjects including arachidonic and linoleic acid (supplement Table 2).

The baseline characteristics of psoriasis patients are listed in Table 1 and after randomization were balanced for age, sex, race, and body mass index according to atorvastatin treatment or not. There was no significant difference in psoriasis severity (median psoriasis area and severity index; 5.6 vs. 4.0, p = 0.69), median LDL-C (111 vs. 108 mg/dL, p = 0.20), and fatty acid composition between groups (Table 1 and Table 2A/2B). After two weeks of high-intensity statin therapy, the median reduction from baseline LDL-C was 44% compared to a non- significant 7% (111 mg/dL, IQR [110 – 121] to 103 mg/dL, IQR [102 – 127] reduction in the no-treatment group (P < 0.0001 for difference between groups). A similar reduction in total and non-HDL cholesterol was seen, without a significant reduction in pro-inflammatory cytokines including high-sensitivity C-reactive protein (data not shown).

Table 1.

Psoriasis Patient Characteristics by Treatment Group.

Characteristics	No Treatment (n = 10)	Atorvastatin (n = 20)	p-value
Age, y	35 (28 – 37)	44 (32 – 52)	0.16
Male sex,%	5 (50)	9 (45)	0.80
Body mass index, kg/m2	28 ± 6	28 ± 7	0.97
Systolic blood pressure	121 ± 10	127 ± 17	0.40
Diastolic blood pressure	75 ± 6	76 ± 12	0.87
Psoriasis
Psoriasis PASI score,	5.6 (3.2 – 19)	4.0 (3.6 – 6.9)	0.69
Psoriatic arthritis	3 (15)	4 (40)	0.13
Biologic therapy,%	4 (40)	14 (70)	0.11
Lipids
Total Cholesterol, mg/dL	179 (178 – 186)	182 (162 – 216)	0.79
Triglyercides, mg/dL	83 (58 – 150)	85 (61 – 104)	0.98
LDL-C, mg/dL	111 (110 – 121)	108 (83 – 131)	0.20
HDL-C, mg/dL	45 (43 – 52)	56 (46 – 63)	0.07

Data are mean ± SD, Median (IQR), or n (%). HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; PASI Score, psoriasis area severity index.

Table 2.

Change in Fatty Acid Composition (%) After Two-Weeks of Atorvastatin or No-Treatment.

Table 2A Fatty acid	No Treatment (n = 10) Baseline	Follow-up	p-value
12:0	0.24 ± 0.24	0.21 ± 0.23	0.11
14:0	0.93 ± 0.34	0.70 ± 0.24	0.02
14:1	0.17 ± 0.11	0.14 ± 0.13	0.11
16:0	21.86 ± 1.97	21.33 ± 1.28	0.49
16:1	1.63 ± 0.36	1.39 ± 0.27	0.06
18:0	7.64 ± 1.34	7.65 ± 1.12	0.99
18:1n-9	18.11±2.17	18.05 ± 2.11	0.9
18:1n-7	1.33 ± 0.33	1.35 ± 0.25	0.85
18:2n-6 (LA)	33.51 ± 4.52	35.15 ± 5.08	0.26
18:3n-6	0.46 ± 0.18	0.4 ± 0.16	0.42
18:3n-3	0.61 ± 0.2	0.56 ± 0.24	0.46
20:0	0.2 ± 0.05	0.18 ± 0.04	0.41
20:1	0.12 ± 0.03	0.11 ± 0.03	0.35
20:2n-6	0.14 ± 0.04	0.18 ± 0.11	0.19
20:3n-6	1.36 ± 0.49	1.33 ± 0.48	0.79
20:4n-6 (AA)	7.88 ± 1.07	7.77±2.17	0.85
20:5n-3	1.27 ± 2.03	0.46 ± 0.17	0.24
22:4n-6	0.17 ± 0.05	0.16 ± 0.05	0.57
24:0	0.16 ± 0.06	0.14 ± 0.05	0.32
22:5n-3	0.48 ± 0.19	0.46±0.17	0.62
22:6n-3	1.6 ± 1.07	1.6 ± 1.11	0.99
Table 2B Fatty acid	Statin (n = 20) Baseline	Follow-up	p-value
12:0	0.27 ± 0.22	0.37 ± 0.3	0.03
14:0	0.88 ± 0.43	0.84 ± 0.5	0.69
14:1	0.17 ± 0.14	0.21 ± 0.18	0.06
16:0	22.29 ± 2.38	21.83 ± 2.27	0.35
16:1	1.64 ± 0.84	1.71 ± 1.00	0.59
18:0	8.07 ± 1.16	8.31 ± 1.32	0.11
18:1n-9	18.50 ± 2.48	20.56 ± 5.08	0.06
18:1n-7	1.39 ± 0.36	1.63 ± 0.35	0.001
18:2n-6 (LA)	33.31 ± 4.84	29.76 ± 4.15	0.002
18:3n-6	0.45 ± 0.24	0.45 ± 0.16	0.95
18:3n-3	0.62 ± 0.19	0.63 ± 0.37	0.84
20:0	0.18± 0.06	0.19 ± 0.09	0.35
20:1	0.14 ± 0.04	0.18 ± 0.09	0.03
20:2n-6	0.18 ± 0.05	0.18 ± 0.04	0.59
20:3n-6	1.29 ± 0.25	1.26 ± 0.25	0.54
20:4n-6 (AA)	7.73 ± 1.38	8.99 ± 2.10	0.007
20:5n-3	0.60 ± 0.35	0.62 ± 0.28	0.88
22:4n-6	0.18 ± 0.04	0.19 ± 0.04	0.2
24:0	0.19 ± 0.13	0.16 ± 0.09	0.13
22:5n-3	0.43 ± 0.08	0.42 ± 0.09	0.84
22:6n-3	1.48 ± 0.53	1.49 ± 0.58	0.84

Data are mean ± SD with fatty acid composition as a percent of the whole. AA; arachidonic acid. LA; Linoleic acid.

For the outcome of this study, 40 mg of atorvastatin therapy lowered LA 12% (33% – > 29%, p < 0.001) and raised AA 14% (7.7 – > 9.0, p < 0.01, Table 2A/2B). No change in LA or AA were noted in the no-treatment group. Sensitivity analyses exploring the composition of LA and AA at study end (supplement Table 3A) and percent change in LA and AA (supplement Table 3B) yielded largely similar results. A correlation between change in LDL-C and LA (r = 0.45, p < 0.01) and opposite change in AA (r = −0.29, p = 0.09) was observed (Fig. 1). Whole blood RNA analysis revealed a significant increase in both fatty acid desaturase (FADS)1 (p = 0.03) and FADS2 (p = 0.02) in the statin treated group as opposed to no significant changes in the no-treatment group (Fig. 1).

Fig. 1.

Relationship between change in low density lipoprotein (LDL) cholesterol, fatty acid composition and FADS transcript expression. (A) Relationship between percent change in LDL and change in arachidonic acid (AA) and linoleic acid (LA) composition (assessed using Pearson’s correlation coefficient). (B) Fatty acid desaturase (FADS) 1 and 2 whole blood transcript expression at baseline and 2 week follow-up after 40 mg of atorvastatin or no-treatment. FADS expression assessed via whole blood paxgene RNA sequencing with transcripts expressed as normalized count values (NCV). Paired t-tests, *<0.05.

4. Discussion

Statins are HMG-CoA reductase inhibitors that reduce circulating LDL-C, reduce ASCVD events, and may also exhibit anti-inflammatory, pleotropic properties. In line with this, our results of decreased LDL-C while enhancing circulating AA are similar to what others show in hypercholesteremic patients without an underlying systemic immune mediated condition. [14]. LA → AA conversion is catalyzed by FADS1 and indirectly enhanced by FADS2. Concordant with this, our findings of peripheral whole blood upregulation of FADS1/2 transcripts in vivo, are similar to what others show in vitro, whereby statins enhance HepG2 FADS1/2 mRNA and protein expression [15] attributed to activation of FADS1 Δ5-desaturation by sterol regulatory element-bindings protein-1. [16]

Most in vivo data are congruent with our findings. In hypercholesterolemic men, simvastatin reduced LA by 5.3% while AA increased 14.2%. [5] Similar results were obtained in hypercholesterolemic men treated with either simvastatin or atorvastatin, with LA reduced 1.65% and AA increased 2.09%. [17] Other studies suggest that when given simvastatin, LA is used for synthesis of AA. [18] AA is the substrate for the production of thromboxane A2 via the cyclooxygenase pathway, which is a platelet aggregator and associated with increased risk of myocardial infarction and stroke. However, AA is a substrate for many other signaling molecules including the lipoxygenase pathway which modulates lipoxins, and multiple other anti-inflammatory pathways [19]. Thus, our findings of increased AA and decreased LA after statin administration do not necessarily imply a deleterious effect on ASCVD outcomes in psoriasis but rather that more AA is available for signaling. Further studies are required to elucidate whether greater substrate availability has disease implications [19].

A sterol response element is located in intron 1 of FAD2 and, in vitro, appears to mediate potentiation of FADS1 by other statins such as simvastatin [20]. Our data are consistent with the hypothesis that the same mechanism upregulates FADS1 and FADS2, and drives a global decrease in LA and increase in AA via enhanced FADS1 desaturation [21,22]. An insertion-deletion polymorphism, rs66698963, nearby this sterol response unit modulates circulating AA levels. These observations indicate that the relative biosynthesis of AA is controlled at the level of FADS1 gene expression.

Omega-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are well known to be antagonistic to the omega-6 AA [23]. One potential clinical implication resulting from our findings is that the circulating AA levels may be dependent upon statins and thus require higher doses of EPA/DHA to achieve a clinical benefit. This hypothesis warrants testing in further clinical-translational studies. Other limitations to our study include that this was an unplanned secondary analyses, with a small sample-size, and potentially underpowered. We also did not measure FADS 1/2 enzyme activity, making clinical applicability of our findings (via a particular mechanism) not yet clear. Therefore, further research is required to understand fatty acid balance and the impact of statins on circulating fatty acids. In conclusion, 40 mg/d of atorvastatin every day for 2 weeks alters long-chain fatty acid metabolism by altering both fatty acid desaturases and reducing plasma levels of LA and increasing AA, in adults with psoriasis.

Abbreviations

AA

Arachidonic acid

CV

Cardiovascular

ASCVD

Atherosclerotic Cardiovascular Disease

DHA

Docosahexaenoic acid

EPA

Eicosapentaenoic acid

FADS

Fatty acid desaturase

LA

Linoleic acid

LDL-C

Low-density lipoprotein cholesterol