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Inflammasome Signaling and Impaired Vascular Health in Psoriasis

Originally publishedhttps://doi.org/10.1161/ATVBAHA.118.312246Arteriosclerosis, Thrombosis, and Vascular Biology. 2019;39:787–798

Abstract

Objective—

Psoriasis is an inflammatory skin disease which heightens the risk of cardiovascular disease. This study directly investigated vascular endothelial health and systemically altered pathways in psoriasis and matched controls.

Approach and Results—

Twenty patients (mean age, 40 years; 50% male) with active psoriasis and 10 age-, sex-matched controls were recruited. To investigate systemically alerted pathways, a deep sequencing omics approach was applied, including unbiased blood transcriptomic and targeted proteomic analysis. Vascular endothelial health was assessed by transcriptomic profiling of endothelial cells obtained from the brachial veins of recruited participants. Blood transcriptomic profiling identified inflammasome signaling as the highest differentially expressed canonical pathway (Z score 1.6; P=1×10-7) including upregulation of CASP5 and interleukin (IL)-1β. Proteomic panels revealed IL-6 as a top differentially expressed cytokine in psoriasis with pathway analysis highlighting IL-1β (Z score 3.7; P=1.02×10-23) as an upstream activator of the observed upregulated proteins. Direct profiling of harvested brachial vein endothelial cells demonstrated inflammatory transcript (eg, IL-1β, CXCL10, VCAM-1, IL-8, CXCL1, Lymphotoxin beta, ICAM-1, COX-2, and CCL3) upregulation between psoriasis versus controls. A linear relationship was seen between differentially expressed endothelial inflammatory transcripts and psoriasis disease severity. IL-6 levels correlated with inflammatory endothelial cell transcripts and whole blood inflammasome-associated transcripts, including CASP5 and IL-1β.

Conclusions—

An unbiased sequencing approach demonstrated the inflammasome as the most differentially altered pathway in psoriasis versus controls. Inflammasome signaling correlated with psoriasis disease severity, circulating IL-6, and proinflammatory endothelial transcripts. These findings help better explain the heightened risk of cardiovascular disease in psoriasis.

Clinical Trial Registration—

URL: http://www.clinicaltrials.gov. Unique identifier: NCT03228017.

Highlights

  • Psoriasis is associated with endothelial inflammatory activation.

  • Inflammasome signaling is highly upregulated in patients with psoriasis.

  • Altered inflammasome signaling is associated with impaired vascular health in patients with psoriasis.

Introduction

Psoriasis is a chronic inflammatory autoimmune skin disease affecting ≈3% of the population in the United States.1 Large population studies suggest psoriasis increases the risk of cardiovascular disease (CVD) upwards of 50%.1–3 Young adults aged under 50 years with severe psoriasis are at even higher CVD risk, with risk of myocardial infarction 2× to 3× that of age-matched controls.4 The dysfunction in psoriasis includes systemic inflammation involving cytokines, such as IL (interleukin)-17, TNF (tumor necrosis factor)-α, and IFN (interferon)-γ.5 Inflammation in active psoriasis has been associated with heightened CVD risk with a notable overlap between expressed cytokines seen in psoriasis and those associated with atherosclerosis.5–9

Impaired vascular health, including endothelial inflammatory activation, is among the initiating factors of atherosclerosis.10 Increased expression of endothelial vascular adhesion molecules, a hallmark of endothelial activation, increases arterial wall translocation of leukocytes into the subendothelium, thus potentiating atherosclerosis.11 Direct assessment of endothelial activation allows insights into the initiation of atherosclerosis in specific disease states.12,13 Indirect assessment of the vasculature suggests that aortic inflammation as detected by [18F]-fluorodeoxyglucose positron emission tomography (FDG-PET) is present in psoriasis.14 However, direct evidence of impaired vascular health and corresponding mechanisms are lacking in patients with psoriasis.

To gain insight into mechanisms of CVD risk in psoriasis, we used a deep sequencing omics approach to integrate circulating blood transcriptomic signatures and a targeted serum proteomic analysis among patients with psoriasis and age-, sex-matched controls. Recruited participants then underwent analysis of harvested brachial vein endothelial cells to directly assess vascular health and the endothelial transcriptome ex vivo. We hypothesized that the endothelium in patients with psoriasis is altered, proinflammatory, and that those alterations correlate with psoriasis disease activity. The platform of integrating blood transcriptional signatures, targeted serum proteomics, and ex vivo endothelial analyses would represent a provisional mechanistic pathway to explain impaired vascular health and hence CVD risk in patients with psoriasis.

Materials and Methods

The authors declare that supporting data are available within the article and its online-only Data Supplement. Additional data that support the findings of this study are available from the corresponding author on reasonable request.

Participant Recruitment

Patients with psoriasis were recruited from New York University Langone Health outpatient clinics between July 2017 and April 2018 as part of an ongoing study investigating vascular health in psoriasis. Active psoriatic disease was confirmed and graded by a board-certified Dermatologist or board-certified Rheumatologist as appropriate (>1% body surface area of plaque psoriasis or >1 swollen/tender joint).

Psoriasis participants were excluded if they were taking aspirin or lipid-lowering therapy or had any major medical comorbidity, including CVD or autoimmune diseases aside from psoriasis or psoriatic arthritis. Control subjects from the community were recruited in a 2:1 (psoriasis to control) fashion. As psoriasis participation accrued, age and sex of the psoriasis participants were averaged and targeted recruitment used to find appropriate matched controls. The study protocol was approved by the New York University School of Medicine institutional review board (17-00692) in line with the Declaration of Helsinki. All subjects provided written informed consent before participation.

Study Visit

Participants were asked to fast >4 hours before the visit. Clinical data, including blood pressure, heart rate, and anthropometric assessments, were collected at the time of the visit by a licensed physician. Next, as previously described and published,15 a 20-gauge angiocatheter was inserted into the brachial forearm. Three J-shaped vascular guidewires (Teleflex, Inc, Reading Pa) were then sequentially advanced into the brachial vein up to 10 cm and washed in dissociation buffer. Blood collection, including serum, plasma, and whole blood RNA was performed after vascular endothelial cell harvesting. Lipid profiles, hs-CRP (high-sensitivity C-reactive protein) and complete blood count were assessed using standard protocols.

Targeted Proteomic Panels

As previously described and reported, aliquots of stored samples were analyzed using the OLINK Proseek multiplex assay Inflammation I, Cardiovascular II/III profiles.16 Briefly, oligonucleotide-labeled antibody probes with proximity extension assay technology bind to their designed target. These antibody pairs attach to their designed target and create a new DNA amplicon. The amplicons were quantified using a Fluidigm BioMark HD real-time polymerase chain reaction platform. Data are reported as Normalized Protein expression, a unit of measurement based on a Log2 scale.

Unbiased Whole Blood 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). Before RNA sequencing, yield, quantity, and quality of the RNA were 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).17 Read count tables were generated using HTSeq (v0.6.0),18 normalized based on their library size factors using DESeq (v3.7),19 and differential expression analysis was performed. Pathway and gene set enrichment analysis was performed using ClusterProfiler R package (v3.6.0).20 All downstream statistical analyses and generating plots were performed in R environment (v3.1.1) (http://www.r-project.org/). An exploratory P value <0.05 was used to determine statistical significance. Ingenuity pathway analysis (Qiagen Bioinformatics, Redwood City, CA) was used to discover differentially expressed pathways.

In Vitro Human Aortic Endothelial Methods and Analysis

HAECs (human aortic endothelial cells) were cultured in Endothelial Cell Growth Medium MV 2 (Promocell GmbH) at passage 3. HAECs were stimulated with PBS, IL-17 (200 ng/mL), IL-17+TNF-α (200+10 ng/mL), IL-17+ IFN-γ (200+20 ng/mL) in duplicate. Cytokine combinations were chosen based on previous investigations by others evaluating in vitro inflammatory endothelial cell response (specifically IL-17+TNF-α) and known prominence (IL-17+IFN-γ) in the pathogenesis of both psoriasis and atherosclerosis.5,21–24 RNA extraction was performed using an RNeasy-MICRO kit (QIAGEN, Redwood City, CA). Gene expression analysis was performed using Affymetrix human transcriptome array 2.0 (Affymetrix, Santa Clara, CA). Data were analyzed through the expression console software 1.3.1 and Affymetrix’ transcriptome analysis console software (Affymetrix, Santa Clara, CA). Genes with a P value <0.05 were considered statistically significant.

Brachial Endothelial Immunostaining

As previously described,13 dissociated endothelial cells were placed in red blood cell lysis buffer then fixed in 10% formaldehyde and dehydrated overnight. Endothelial cells were permeabilized with 0.1% Triton X-100 (Acros Organics), blocked with 4% FBS, and incubated 4 hours with CD144 (VE-cadherin [vascular endothelial cadherin]) goat anti-human antibodies (1:20; R&D systems) along with 488-conjugated donkey anti-goat secondary antibodies (1:50; Jackson ImmunoResearch). Nuclear fluorescence of NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) was identified by targeting the p65 subunit with rabbit anti-human antibodies (1:50; Millipore Sigma) and secondary staining with 594-conjugated donkey anti-rabbit antibodies (1:50; Jackson ImmunoResearch). Nuclei were identified through 4’,6-diamidino-2-phenylindole staining. Staining was visualized with UV light under an AxioObserver.Z1 fluorescent microscope (Zeiss, Oberkochen, Germany). Negative controls were generated with their appropriate antibodies (Methods in the online-only Data Supplement). Imaging processing and analysis for p65 NFκB nuclear translocation was performed using ImageJ (National Institutes of Health). Endothelial cells with both cellular and nuclear integrity were assessed. The percentage of p65 NFκB/4’,6-diamidino-2-phenylindole colocalization was calculated using the JACoP plug-in of ImageJ (National Institutes of Health) after appropriate thresholds were established.25

Brachial Endothelial Transcript Analysis

Brachial endothelial cell pellets were resuspended in isolation buffer, incubated with biotinylated mouse anti-human monoclonal antibody directed against CD146 (1:200; Millipore Sigma), and isolated with streptavidin magnetic FlowComp Dynabeads (1:100). Endothelial cells underwent mRNA extraction using RNAqueous–micro RNA isolation kit (Invitrogen, Carlsbad CA). mRNA was converted to cDNA (Quantbio, Beverly, MA) and amplified via PerfeCta PreAmp SuperMix (Quantabio). TaqMan (Life Technologies) primers were used on an Applied Biosystems 7500 Fast Real-Time PCR System (Foster City, CA). To ensure reproducibility across analysis, results are represented as normalized to hARP (human acidic ribosomal protein) for each sample and gene.26

Immunohistochemistry and Immunofluorescence of Human Skin

Frozen tissue sections of skin biopsies were blocked with 10% normal goat serum for 20 minutes and then incubated with Anti-NFκB p65 antibody (Millipore Sigman) overnight. Biotin-labeled goat anti-rabbit (Vector Laboratories) was used to detect the NFκB antibody. The staining signal was amplified with avidin-biotin complex (Vector Laboratories) and developed using chromogen 3-amino-9-ethylcarbazole (Millipore Sigma).

For immunofluorescence, skin samples were fixed with acetone and blocked in 10% normal chicken serum (Vector Laboratories) for 30 minutes. The skin tissue sections were incubated with NFκB p65 rabbit antibody (Millipore Sigma) and VE-cadherin polyclonal goat antibody (R&D systems) overnight at 4°C. The next day, the tissues were amplified with chicken anti-rabbit Alexa Fluor 594 (Invitrogen) and chicken anti-goat Alexa Fluor 488 (Invitrogen) for 30 minutes. Negative controls were generated with their appropriate antibodies (Methods in the online-only Data Supplement).

Images were acquired using the appropriate filters of a Zeiss Axioplan 2 widefield fluorescence microscope with a Plan Neofluar 20×0.7 numerical aperture lens and a Hamamatsu Orca ERcooled charge-coupled device camera, controlled by METAVUE software (MDS Analytical Technologies, Downington, PA). Images in each figure are presented both as single color stains (green and red) located above the merged image so that localization of 2 markers on similar or different cells can be appreciated. Cells that coexpress the 2 markers in a similar location are yellow in color. A white line denotes the junction between the epidermis and the dermis

Statistical Analysis Comparing Psoriasis to Control Participants

Continuous data are presented as mean±SD or median (interquartile range) as appropriate. Categorical data are presented as total number (percentages). Normally distributed continuous variables were assessed through a Student t test while nonnormally distributed continuous variables were assessed through Wilcoxon rank-sum test. Categorical variables were assessed through χ2 analysis. Linear regression was used to evaluate the association between psoriasis severity and outcome transcripts with multivariable analysis to account for factors in the American Heart Association/American College of Cardiology 2013 pooled cohort CVD risk score. Regression analysis data are reported as β-coefficient (95% CI) and P value. A P value <0.05 was considered statistically significant. All analyses were performed in STATA v.14 (College Station, TX: StataCorp LP). The authors had full access to all data in this study and take responsibility for its integrity and the data analysis.

Sample Size

Studies in inflammatory populations have noted 2- to 3-fold differences (disease versus control) in endothelial inflammatory activation.27 Aortic vascular inflammation has been noted to be upwards of 13% higher in psoriasis compared with control.28 Based on these previous studies and our own in vitro cytokine-stimulated HAEC work, we hypothesized to see at least a 2-fold increase between transcript expression in psoriasis compared with controls. A target analysis goal of n=20 for psoriatic disease and n=10 for controls gave us >80% power to detect a baseline endothelial difference of 2× between groups with an alpha level of 0.05 (G*Power 3.1.9.2).

Results

Clinical Characteristics of Recruited Subjects

This study sought to characterize vascular health in patients with psoriasis. Thirty participants were evaluated: 20 psoriasis and 10 matched controls. Demographics and clinical characteristics are presented in Table 1. Age, sex, race/ethnicity, body mass index, and blood pressure were similar between groups. Cardiovascular risk, measured using the American College of Cardiology/American Heart Association pooled cohort equation,29 was low and not different between groups (psoriasis 4.0±6.2% versus controls 4.1±6.6%; P=0.97).

Table 1. Clinical Characteristics

CharacteristicsControl (N=10)Psoriasis (N=20)P Value
Age, y, median (IQR)42.5 (30–58)39.5 (34.5–53)0.71
Male sex, %5 (50)10 (50)1
White, %6 (60)10 (50)0.58
Body mass index, kg/m228±328±70.52
Hypertension, %0 (0)2 (6)
Systolic blood pressure, mm Hg126±16138±140.84
Diastolic blood pressure, mm Hg73±976±100.54
Type 2 diabetes mellitus, %0 (0%)1 (3)
Current tobacco use, %0 (0%)1 (3)
ACC/AHA ASCVD risk score,* %4.0±6.24.1±6.60.97
Psoriasis
 Disease duration, y, median (IQR)15 (10–22.5)
 Psoriatic arthritis, %7 (35)
 BSA, %, median (IQR)6 (3.5–10)
 PASI score, median (IQR)5.5 (3.8–14.1)
 Biologic therapy, %11 (55)
 Any other systemic therapy, %6 (30)
 Light therapy, %6 (30)

Data are mean±SD or N (%) unless otherwise stated. ACC indicates American College of Cardiology; AHA, American Heart Association; ASCVD, atherosclerotic cardiovascular disease; BSA indicates body surface area of psoriasis; IQR, interquartile range; and PASI, psoriasis area severity index.

*ACC/AHA Atherosclerotic Cardiovascular Risk Score.

Participants with psoriasis had on average 15 years of disease duration with a median body surface area of 6% (interquartile range, 3.5–10) psoriasis plaque and median PASI (psoriasis area severity index) score of 5.5 (3.8–14.1) consistent with a cohort of mild to moderate active psoriasis at the time of enrollment. Seven patients (35%) had concomitant psoriatic arthritis (Table 1). Hs-CRP trended higher in psoriasis versus controls (1.8 mg/L; interquartile range; 0.5–1.4 versus 0.80 mg/L interquartile range: 0.7–4.1, respectively, P=0.15; Table 2).

Table 2. Laboratory Characteristics

Laboratory ParametersControl (N=10)Psoriasis (N=20)P Value
Hematologic studies
 WBC, ×103 cells/mm35.9±17.5±30.34
 Hematocrit, %40.0±438.8±50.44
 Platelets, cells/L256±75258±831.0
 Mean platelet volume, fL9.2±29.3±10.52
 Absolute neutrophils, ×103 cells/mm33.7±1.34.8±2.80.57
 Absolute monocytes, ×103 cells/mm30.36±0.20.52±0.20.17
 Neutrophil:lymphocyte ratio2.2±12.5±10.78
Serum laboratory measurements
 hs-CRP, mg/L, median (IQR)0.80 (0.5–1.4)1.8 (0.7–4.1)0.15
 Total cholesterol, mg/dL154±24175±480.09
 Triglycerides, mg/dL, median (IQR)62 (56–83)83 (55–131)0.30
 LDL cholesterol, mg/dL90±22105±410.49
 HDL cholesterol, mg/dL49±952±160.64
 IL-17A, median (IQR)*0.9 (0.4–1.8)2.1 (1.0–5.0)<0.01
 IL-6, median (IQR)*3.9 (3.5–4.6)4.5 (3.9–5.5)0.03

Data are mean±SD or N (%) unless otherwise stated. HDL indicates high-density lipoprotein; hs-CRP, high-sensitivity C-reactive protein; IL, interleukin; IQR, interquartile range; LDL, low-density lipoprotein; NPX, normalized protein expression; and WBC, white blood cell.

*Data reported as NPX (NPX−log2 scale, entire cohort assessed n=15 controls, n=22 psoriasis).

Transcriptome Profiling of Blood Using RNA Sequencing Reveals an Inflammatory Signature in Psoriasis

High throughput sequencing technologies are a valuable platform to investigate complex and dynamic disease processes. To further our understanding of a heightened inflammatory state in psoriasis, transcriptomics through unbiased whole blood RNA sequencing was performed. Overall, 758 transcripts were differentially expressed (P<0.05; 430 upregulated and 328 downregulated) between 10 psoriasis and 10 age- and sex-matched controls (Figure 1A and 1B). Ingenuity pathway analysis demonstrated inflammasome signaling to be the highest differentially expressed canonical pathway (Z score 1.6; P=1×10-7; Figure I in the online-only Data Supplement). There was significant overexpression of CASP5, SOCS3, TNF, and IL-1β (Figure 1B and 1C). Predicted top regulators of differentially expressed transcripts were signaling proteins and transcription factors known to be elevated in psoriasis including IFN-γ, IL-6, IL-4, NFκB, and IL-12 (Table I in the online-only Data Supplement).5 Biological process analysis revealed substantial pathway activation in inflammatory response, cell-to-cell signaling and interaction, along with lipid metabolism and cardiovascular system development (atherosclerosis and immune function related pathways, Figure 1D). To better understand the clinical relevance of the transcripts identified from blood RNA sequencing, we investigated their association with psoriasis severity and noted a positive correlation between the top 20 upregulated blood transcripts and psoriasis disease severity (Table II in the online-only Data Supplement). Collectively, these data demonstrate the inflammasome as the most differentially altered pathway in psoriasis (versus controls) which correlates with psoriasis disease activity.

Figure 1.

Figure 1. Enrichment in inflammatory- and atherosclerosis-associated pathways in the blood transcriptome of patients with psoriasis. A, Heat map of 758 differentially expressed transcripts between 10 psoriasis and 10 age-, sex-matched controls as determined by blood RNA sequencing (P<0.05). B, Volcano plots of transcripts in whole blood from participants with psoriasis vs controls. The y axis corresponds to the P value (−log10), and the x axis displays the log2 fold change. The red dots represent the significantly upregulated transcripts, and blue dots significantly downregulated transcripts (P<0.05). C, Differential expression of IL (interleukin)-1β, TNF (tumor necrosis factor)-α, and the top 2 upregulated transcripts (CASP5 [caspase 5], SOCS3 [suppressor of cytokine signaling 3]) in patients with psoriasis compared with controls. D, Pathway analysis (ingenuity pathway analysis) of differentially regulated transcripts highlights the inflammatory, immune response, and cardiovascular disease biological process which are upregulated in psoriasis over control subjects P<0.05*, P<0.01**, P<0.001***. FPKM indicates fragments per kilobase of transcript per million mapped reads.

Proteomics in Psoriasis Reveal an Inflammatory Signature

Protein panels revealed plasma levels of IL-17A and IL-6 as the top 2 differentially expressed cytokines in psoriasis compared with controls (Table 2). Consistent with findings in other psoriasis cohorts, serum levels of inflammatory proteins, including IL-6, but not IL-17A, correlated with psoriasis disease activity16 (Table III in the online-only Data Supplement). Ingenuity pathway analysis revealed the top 3 predicted upstream regulators of protein expression were TNF (Z score 4.2; P=2.9×10-24), IL-1β (Zscore 3.7; P=1.02×10-23), and IFN-γ (Z score 2.8; P=2.4×10-19). Finally, the top differentially expressed canonical pathway was HMGB1 (high mobility group box 1; Z score 2.6; P=1.2×10-8, data not shown), a known regulator of inflammatory gene expression and a pathway induced by inflammasome activation.30,31

Inflammatory Psoriasis Profiles Associate With Activated Endothelial Cells

Systemic inflammation in psoriasis associates with heightened CVD risk.6 However, the mechanisms underlying this risk are not entirely understood. Endothelial cells act as a first responder to inflammatory cytokines and are involved in the early pathogenesis of atherosclerosis.32 To model how psoriasis inflammatory processes relates to vascular health, endothelial cells were assessed in vitro in response to implicated cytokines from our proteomic studies, each of which are also involved in the pathogenesis of psoriasis (IL-17, TNF-α, IFN-γ).21–23 Unbiased microarray analysis of HAECs stimulated with IL-17+IFN-γ compared with PBS revealed 3904 differentially expressed genes (P<0.05; 1657 upregulated; 2247 downregulated), and IL-17+TNF-α compared with PBS revealed 4687 differentially expressed genes (P<0.05; 2155 upregulated, 2532 downregulated, data not shown). Pathway analysis revealed NFκB signaling as a key regulator of the cytokine-stimulated HAEC inflammatory response (Figure II in the online-only Data Supplement). Additionally, in canonical pathway analysis, inflammasome signaling was highly upregulated after IL-17+IFN-γ stimulation (Z score 2.3; P=1.1×10-5). Finally, biological pathway analysis of the psoriasis blood transcriptome along with in vitro transcriptomics of cytokine-stimulated HAECs revealed overlapping biological processes related to atherosclerosis and inflammation (Figure III in the online-only Data Supplement). In summary, the blood transcriptome, proteome, and in vitro endothelial transcriptome all suggested a connection between psoriasis disease activity, inflammasome signaling, and markers of endothelial inflammation and activation.

Inflammatory Transcriptomic Signature Is Present in Endothelial Cells Harvested From Patients With Psoriasis

To directly investigate the endothelium in psoriasis, we harvested primary brachial venous endothelial cells from psoriasis and controls. Candidate transcripts were evaluated based on cytokine stimulation HAEC studies (Figure 2A) and through existing literature describing endothelial inflammatory activation (VCAM-1, ICAM-1, MCP-1, CCL3, COX-2, VWF, VEGFA)33,34 as well as transcripts observed in the pathophysiology of psoriasis (CXCL10, CX3CL1, IL-8, CXCL1, Lymphotoxin beta, IL-1β.35–37 Transcriptome profiling of harvested brachial vein endothelial cells was positive for the endothelium-specific marker VE-cadherin and revealed no difference between groups in VE-cadherin or housekeeping genes hARP or β-actin (Table IV in the online-only Data Supplement). Comparing psoriasis with controls, we found significant elevations in IL-1β (3-fold), IL-8 (10-fold), CCL3 (8-fold), COX-2 (3-fold), and Lymphotoxin beta (3.5-fold; P<0.05; Figure 2B).

Figure 2.

Figure 2. Endothelial cells from psoriasis patients reveal inflammatory activation. A, Differential transcript expression after in vitro human aortic endothelial cell stimulation with IL (interleukin)-17 vs PBS, IL-17+TNF (tumor necrosis factor)-α vs PBS, IL-17+IFN (interferon)-γ versus PBS (P<0.05 for all changes). B, Direct analysis of venous endothelial cells harvested from patients with psoriasis compared with controls show transcript upregulation in intracellular adhesion molecules (VCAM-1, ICAM-1), inflammation (COX-2), as well as chemokines (CXCL10, CXCL1, CX3CL1, CCL3, MCP-1) interleukins and TNFs (IL-1β, IL-8, Lymphotoxin beta). C, Differential endothelial transcript expression in psoriasis over controls stratified by psoriasis severity (control, psoriasis area, and severity index [PASI]: ≤10, >10). D, Endothelial inflammatory activation in patients with psoriasis, as assessed by nuclear p65 NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) localization in the vascular endothelium of brachial vein endothelial cells. Direct immunofluorescence staining of harvested venous endothelial cells (VE-cadherin [vascular endothelial cadherin], green) of patients with psoriasis compared with healthy controls show increased p65 NFκB (red) nuclear (4’,6-diamidino-2-phenylindole [DAPI]–blue) colocalization (n=6). P<0.05*, P<0.01**. VEGFA indicates vascular endothelial growth factor A; and VWF, von Willebrand factor.

Significant associations were also noted between psoriasis disease severity and many of the upregulated endothelial transcripts (Figures 2C and 3, Table 3, Figure IV in the online-only Data Supplement). After adjustment for traditional cardiovascular risk factors (age, sex, systolic blood pressure, total cholesterol, HDL [high-density lipoprotein] cholesterol, diabetes mellitus, smoking) and systemic psoriasis biologic therapy, the associations between psoriasis disease severity and inflammatory endothelial transcripts remained significant. Analysis of psoriasis disease severity using body surface area yielded similar findings (Table V and Figure V in the online-only Data Supplement).

Figure 3.

Figure 3. Representative regression plot showing a linear relationship between psoriasis disease severity (psoriasis area and severity index) and inflammatory transcript expression. VCAM (vascular cell adhesion molecule)-1, r=0.81, P<0.001; CXCL10 (C-X-C motif chemokine 10), r=0.89, P<0.001.

Table 3. Association Between Psoriatic Disease Severity and Inflammatory Endothelial Transcripts by PASI Score

Gene TranscriptUnivariable*MultivariableP Value
β (95% CI)β (95% CI)
Lymphotoxin beta1.22 (0.47 to 1.96)1.19 (0.47 to 1.92)<0.01
ICAM-10.48 (0.22 to 0.75)0.46 (0.26 to 0.66)<0.001
VCAM-11.42 (0.90 to 1.93)§1.44 (0.91 to 1.96)<0.001
MCP-10.21 (0.09 to 0.32)0.20 (0.02 to 0.38)0.03
CCL30.08 (−0.10 to 0.19)
CX3CL10.08 (−0.39 to 0.56)
CXCL10.08 (−0.002 to 0.17)
CXCL101.89 (1.41 to 2.37)§1.83 (1.15 to 2.52)<0.001
COX-20.32 (−0.12 to 0.77)
IL-8−0.06 (−0.77 to 0.64)
IL-1β1.01 (0.34 to 1.68)0.93 (0.28 to 1.58)0.01

Psoriatic disease severity assessed by PASI. HDL indicates high-density lipoprotein; and PASI, psoriasis area severity index.

*Data on table represented as regression coefficients.

†Multivariable model adjusted for age, sex, systolic blood pressure, total cholesterol, HDL cholesterol, diabetes mellitus, smoking, and biologic therapy.

P<0.01.

§P<0.001.

NFκB Is Activated in Psoriasis Endothelial Cells

To investigate the clinical significance of our blood transcriptomic (Table II in the online-only Data Supplement) and in vitro HAEC experiments (Figure II in the online-only Data Supplement), we performed direct ex vivo p65 NFκB staining of the subcutaneous vascular endothelium in skin biopsies and harvested brachial vein endothelial cells. Subcutaneous psoriasis lesional skin (psoriatic plaque) and nonlesional skin biopsies from patients with psoriasis, compared with skin biopsies from healthy controls, demonstrated increased endothelial cell staining of p65 NFκB (Figure 4A through 4C). Consistent with data in the subcutaneous tissue and cytokine-stimulated HAEC pathway analysis (Figure II in the online-only Data Supplement), nuclear translocation of p65 NFκB in the harvested vascular endothelial cells was greater in psoriasis than in control (Figure 2D).

Figure 4.

Figure 4. Skin biopsies reveal vascular subcutaneous endothelial inflammation is present in patients with psoriasis. A, Immunohistochemistry of p65 NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) in lesional skin, nonlesional skin from a psoriasis patient, and normal skin from a healthy control. B, Immunofluorescence staining of skin biopsies show increased p65 NFκB (red) expression in the vascular endothelium (VE-cadherin [vascular endothelial cadherin], green) in both lesional skin and nonlesional skin of a psoriatic patient compared with normal healthy skin. Colocalization of p65 NFκB and VE-cadherin (yellow). C, Increased magnification of images in B.

Inflammasome Signaling May Be Linked to the Endothelial Inflammatory Transcriptome

Finally, we explored associations between the blood transcriptome, serum proteome signatures, and the vascular endothelium. As noted above, pathway analysis demonstrated inflammasome signaling as the top upregulated canonical pathway (Figure I in the online-only Data Supplement). The NFκB signaling pathway (regulator in psoriasis; Table I in the online-only Data Supplement) regulates the transcription of genes encoding components of the inflammasome and mediates the induction of inflammatory cytokines, such as IL-1β, leading to downstream IL-6 production, thereby contributing to the initiation and development of inflammatory diseases.38,39 In our proteomic data, IL-17A and IL-6 were the 2 highest differentially expressed cytokines (Table 2). Serum levels of both IL-17A and IL-6 correlated with many of the top differentially expressed blood RNA sequencing transcripts. However, serum IL-17A only correlated with the ex vivo endothelial transcript CXCL10 (C-X-C motif chemokine 10; Table VI in the online-only Data Supplement). In contrast, serum IL-6 levels correlated with many differentially expressed ex vivo endothelial cell transcripts directly harvested from the brachial vein (Table 4). Altogether, these data suggest an important link between inflammasome (IL-1β/IL-6) signaling and endothelial activation, a major precursor to atherosclerosis.40

Table 4. Correlation Between Serum IL-6 Levels and Blood and Endothelial Transcripts

TranscriptCorrelationP Value
Blood transcripts
IL-1β*0.60<0.001
NFKB1*0.68<0.001
CASP5*0.77<0.001
MYD88*0.73<0.001
NAIP*0.63<0.01
SOCS30.77<0.001
CD140.77<0.001
MAPK110.59<0.01
IRS10.47<0.05
IL-1RN0.65<0.01
TNF0.460.07
SRF0.570.01
Endothelial ex vivo transcripts
Lymphotoxin beta0.34<0.05
ICAM-10.38<0.05
VCAM-10.66<0.001
MCP-10.51<0.01
CCL30.320.08
CX3CL10.000.99
CXCL10.200.26
CXCL100.80<0.001
COX-20.020.89
IL-8−0.040.81
IL-1β0.240.18

Correlation: Pearson correlation coefficient r assessing the correlation between serum IL-6 levels and inflammatory-related transcripts. Whole blood transcriptome evaluated as FPKM values. Endothelial transcripts were obtained from freshly harvested endothelial cells obtained from the Brachial vein. FPKM indicates fragments per kilobase of transcript per million mapped reads; and IL, interleukin.

*Inflammasome determined by ingenuity pathway analysis.

†IL-6 signaling determined by ingenuity pathway analysis.

Discussion

In this study, unbiased whole blood transcriptomic and targeted proteome analysis pointed to systemic inflammasome signaling as the top differentially expressed pathway. Direct ex vivo analysis of harvested endothelial cells revealed differentially expressed inflammatory endothelial transcripts between psoriasis and control with strong associations observed between psoriasis disease severity, systemic inflammasome signaling, and endothelial inflammatory activation. Finally, NFκB, a known transcription factor implicated in inflammasome signaling,41 was enhanced in psoriasis subcutaneous skin and brachial venous endothelial cells. Altogether, we directly characterized impaired vascular health using ex vivo and in vitro techniques and described altered pathways in psoriasis that contribute to early CVD.6,7,11,23

Endothelial inflammatory activation is among the early vascular abnormalities in the development of atherosclerosis.10,42 The vascular endothelium is the key regulator of vascular system homeostasis, including maintenance of vasomotor tone, regulation of cellular trafficking and adhesion, and thromboresistance.10 In a disease state, activated endothelium express proinflammatory cytokines and adhesion molecules which attract further inflammatory cells, contributing to and perpetuating the development of atherosclerosis.42,43 Among patients with psoriasis and low CVD risk (as noted by a mean cardiac risk score of 4%; Table 1), we demonstrated impaired endothelial health both in vitro and ex vivo and identified upregulated inflammatory transcripts in psoriasis, each of which has previously been implicated in the pathogenesis of atherosclerosis.42,44,45

Prior studies have shown that psoriasis immune-mediated inflammation is associated with aortic inflammation as assessed by FDG-PET.28,46 However, no study has directly investigated the vascular endothelium and systemically altered pathways in an unbiased manner to explore how psoriasis relates to CVD. In our analysis, the majority of harvested endothelial transcripts assessed ex vivo correlated with psoriasis disease severity. The findings described in our study suggest that expression of the cytokines MCP-1, CXCL10, IL-1β, theTNF Lymphotoxin beta and vascular adhesion molecules, ICAM-1, and VCAM-1 may, in part, explain FDG-PET imaging studies describing the association between psoriasis disease severity and large vessel vascular inflammation.14,28,36,37,46–49 However, this concept would need to be investigated in future studies.

Despite the known connection between psoriasis disease severity and vascular inflammation,14,49,50 we observed that the transcripts IL-8, COX-2, and CCL3, were upregulated in psoriasis compared with controls, yet not associated with psoriasis disease activity. Epidemiological studies support the concept that while psoriasis disease severity influences CVD risk, a residual risk of CVD may remain even in those with mild disease.51 Through direct analysis of harvested endothelial cells, our study suggests potential pathways through which CVD risk exists independent of psoriasis disease activity. Finally, the findings that cytokines upregulated in psoriasis lesional skin biopsies21,35,52,53 are also present in large vessel endothelium highlights the systemic inflammatory nature of psoriasis affecting organs beyond the skin.

To expand on the significance of psoriasis endothelial inflammatory activation, we used whole blood transcriptomics and targeted serum proteome analysis to perform a deep sequencing omics approach to inflammation in psoriasis and correlate this with impaired vascular health. In our study, unbiased analysis revealed inflammasome signaling as the top activated canonical pathway in psoriasis which correlated with IL-6 protein levels. The inflammasome is a multimeric protein complex involved in the immune response whose end products including IL-1β and IL-18 along with downstream production of IL-6.40,54CASP5, our top upregulated whole blood transcript, encodes caspase 5, which is present within the inflammasome and activates caspase 1, the enzyme required to cleave pro–IL-1β and pro–IL-18 into active forms.55 Others have noted caspase 5 and inflammasome pathway upregulation in psoriasis plaque keratinocytes.56,57 Therefore, our findings of a circulating inflammasome transcriptomic signature in psoriasis are not surprising and suggest that mechanisms driving skin pathology in psoriasis also drive systemic pathology in psoriasis, such as vascular endothelial inflammation.56,58

In our analysis, serum IL-17A, a known key pathogenic cytokine in psoriasis,5 correlated only with the ex vivo endothelial transcript CXCL10 and not others (Table VI in the online-only Data Supplement). CXCL10 is a known chemoattractant and found at all stages of atherosclerotic development in addition to lesional psoriasis skin.37,59 Notably, this cytokine was also highly differentially expressed after in vitro HAEC analysis and strongly correlated to psoriasis disease severity. Given the controversial role of IL-17A in promoting atherosclerosis,60 our data suggest that CXCL10, IL-17A, and their relationship to CVD risk in psoriasis deserves future investigation.37,61 However, ultimately, the question of inhibiting IL-17A to improve vascular health in psoriasis will need to be tested such as in the ongoing VIP-S trial (Vascular Inflammation in Psoriasis Trial - Secukinumab; http://www.clinicaltrials.gov. Unique identifier: NCT02690701).

Overall, studies of therapies to improve CVD risk in psoriasis have primarily used psoriasis disease–specific medications,5,16,62,63 and our findings suggest that inflammatory mediators which may not necessarily drive the maintenance phase of psoriasis lesional skin pathogenesis,5 strongly correlate with impaired vascular health. The inflammasome IL-1β/IL-6 axis correlated more strongly than IL-17A to ex vivo endothelial upregulated transcripts, a finding which may allow for improved development and evaluation of therapies to reduce CVD risk. Psoriasis preferentially increases the relative risk of CVD in the young, an area where the evidence for primary cardiac prevention therapies are lacking.64 Furthermore, randomized clinical studies assessing biologic therapy in psoriasis (TNF-α inhibitors) to reduce vascular inflammation using in vivo imaging techniques have yielded discrepant results.62,63 Studies have shown that progression of carotid plaque and aortic inflammation are reduced after TNF-α inhibitor therapy.62 However, a recent randomized trial of TNF-α inhibitor therapy in psoriasis improved skin disease and circulating markers of systemic inflammation but failed to improve aortic vascular inflammation assessed by FDG-PET.63

By characterizing the vascular endothelium and linking this to differentially expressed systemic pathways in psoriasis, we have identified promising surrogate endpoints to evaluate the impact of psoriasis specific treatment on CVD risk as the endothelium may be more responsive to changes in circulating cytokines than FDG-PET vascular imaging. Additionally, the inflammasome (IL-1β/IL-6 axis) has been implicated in the pathogenesis of CVD and a recent randomized clinical trial investigating IL-1β inhibition has demonstrated a reduction in CVD events.40,65 Thus, a logical hypothesis emerges that targeting inflammasome signaling in psoriasis is a more precise approach to reduce CVD risk in patients with psoriasis.

Limitations

The primary goal of this study was to directly investigate mechanisms of impaired vascular endothelial health in psoriasis. To do this, because of practical and logistical reasons, we studied freshly harvested venous as opposed to arterial endothelial cells. Direct harvesting of venous endothelial cells to describe molecular mechanisms of endothelial activation is a well-validated innovative technique.12,13,15,66 Although venous and arterial endothelium exhibit different gene expression characteristics,67,68 our findings and approach to studying endothelial activation was supported by in vitro HAEC analysis. We also confirmed that endothelial inflammation (as assessed by p65 NFκB) is present both in the venous (Figure 2D) and arterial (Figure 4) endothelium. Finally, the use of whole blood PAXgene to assess the systemic inflammatory response may limit complete mechanistic insight as this technique does not allow identification of the specific cell types which are responsible for the observed systemic inflammatory changes. However, in support of our findings, the whole blood transcriptomic and serum proteomic findings are supported by an extensive literature investigating inflammatory mediators present in psoriasis plaque.5,16,35–37

There are several other limitations to this current study. The cross-sectional study design does not permit causal inference based on the observed associations of endothelial inflammation, activation, and severity of psoriasis and by itself does not inform on the potential impact on future CVD risk. Additionally, this study may actually underrepresent systemic and endothelial inflammation in psoriasis as the median PASI score was only 5.5, signifying mild to moderate disease but similar to other studies investigating CVD risk in psoriasis.69 Finally, half of patients with psoriasis were receiving immune modulating therapies which has been shown to reduce CVD risk associated serum proteins.16

Conclusions

In conclusion, in patients with psoriasis considered to be at low cardiovascular risk, we show direct evidence of impaired vascular health. Using an omics approach, both blood transcriptomic and targeted proteomic analysis implicate inflammasome signaling as the major circulating inflammatory signature in patients with psoriasis. Psoriasis disease severity and inflammasome signaling each correlated with the observed upregulated inflammatory endothelial transcripts suggesting that the inflammasome signaling pathway may mediate the observed impaired vascular endothelial health and increased CVD risk in psoriasis. These findings have future implications for mechanistic studies assessing CVD risk in psoriasis and highlight several therapeutic pathways which may be a target in future trials to reduce CVD risk in psoriasis.

Nonstandard Abbreviations and Acronyms

CVD

cardiovascular disease

FDG-PET

[18F]-fluorodeoxyglucose positron emission tomography

HAEC

human aortic endothelial cells

hARP

human acidic ribosomal protein

HDL

high-density lipoprotein

hs-CRP

high-sensitivity C-reactive protein

IFN

interferon

IL

interleukin

NFκB

Nuclear factor kappa-light-chain-enhancer of activated B cells

PASI

psoriasis area severity index

TNF

tumor necrosis factor

Acknowledgments

We thank Xuan Li for training in ex vivo endothelial quantitative polymerase chain reaction analysis, Alireza Khodadadi-Jamayran for RNA sequencing analysis, Memet Emin for instruction in endothelial harvesting procedures, Charissa Mia Salud for help in skin immunostaining, and the nursing staff at the NYU Langone Health phototherapy clinic and NYU Clinical and Translational Science Institute.

Footnotes

The online-only Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/ATVBAHA.118.312246.

Correspondence to Michael Garshick, MD, MS, Center for the Prevention of Cardiovascular Disease, New York University Langone Health, 435 E 30th St, 7th Floor, New York City, NY 10016, Email
Jeffrey S. Berger, MD, MS, Center for the Prevention of Cardiovascular Disease, New York University Langone Health, 435 E 30th St, 7th Floor, New York City, NY 10016, Email

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