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Patients with psoriasis have increased risk of cardiovascular disease (CVD) independent of traditional risk factors. The molecular mechanisms underlying the psoriasis-CVD connection are not fully understood. Advances in high-throughput molecular profiling technologies and computational analysis techniques offer new opportunities to improve the understanding of disease connections.
We aim to characterize the complexity of cardiovascular risk in patients with psoriasis by integrating deep phenotypic data with systems biology techniques to perform comprehensive multiomic analyses and construct network models of the two interacting diseases.
The study aims to include 120 adult patients with psoriasis (60 with prior atherosclerotic CVD and 60 without CVD). Half of the patients are already receiving systemic antipsoriatic treatment. All patients complete a questionnaire, and a medical interview is conducted to collect medical history and information on, for example, socioeconomics, mental health, diet, and physical exercise. Participants are examined clinically with assessment of the Psoriasis Area and Severity Index and undergo imaging by transthoracic echocardiography, 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG-PET/CT), and carotid artery ultrasonography. Skin swabs are collected for analysis of microbiome metagenomics; skin biopsies and blood samples are collected for transcriptomic profiling by RNA sequencing; skin biopsies are collected for immunohistochemistry; plasma samples are collected for analyses of proteomics, lipidomics, and metabolomics; blood samples are collected for high-dimensional mass cytometry; and feces samples are collected for gut microbiome metagenomics. Bioinformatics and systems biology techniques are utilized to analyze the multiomic data and to integrate data into a network model of CVD in patients with psoriasis.
Recruitment was completed in September 2020. Preliminary results of 18F-FDG-PET/CT data have recently been published, where vascular inflammation was reduced in the ascending aorta (
This systems biology approach with integration of multiomics and clinical data in patients with psoriasis with or without CVD is likely to provide novel insights into the biological mechanisms underlying these diseases and their interplay that can impact future treatment.
DERR1-10.2196/28669
Psoriasis is a chronic inflammatory disease affecting 2% to 3% of the adult population and is associated with an increased risk of developing other inflammatory diseases, such as cardiovascular disease (CVD), inflammatory bowel disease, and diabetes [
Basic, translational, and clinical research directed separately at psoriasis or CVD has led to the identification of the molecular disease mechanisms and the development of new therapies aimed at psoriasis or CVD. However, this traditional paradigm is built on studies that do not capture deep phenotypes of individual patients, and to identify the central drivers of diseases and enable precision therapy, systems biology–oriented approaches are needed that seek to integrate all relevant available data. Advances in high-throughput molecular profiling and computational analysis techniques offer such novel opportunities to improve the understanding of disease connections and accelerate the discovery of new therapeutic strategies [
Overview of study examinations and analyses. 18F-FDG-PET/CT: 18F-fluorodeoxyglucose positron emission tomography/computed tomography; CVD: cardiovascular disease; CyTOF: cytometry by time-of-flight; PEA: proximity extension assay; UPLC-MS/MS: ultra-high performance liquid chromatography/tandem mass spectrometry.
This study aims to investigate the complex multiscale interactions that drive cardiovascular risks in individual patients with psoriasis, involving specific gene transcripts, protein and lipid markers, signaling pathways, immune cell types, organ systems, and microbiota. Moreover, the study will examine multiscale differences across patient groups with psoriasis having distinct phenotypic traits, for example, with or without CVD, systemic antipsoriatic treatment or psoriatic arthritis, and early or late onset of psoriasis.
The study will include 120 patients (aged ≥30 years) with moderate-to-severe plaque psoriasis, with 60 having prior (over 6 months before inclusion) atherosclerotic CVD, including myocardial infarction, coronary revascularization, ischemic stroke, and/or peripheral artery disease, and 60 not having this history. Furthermore, half of the patients receive systemic antipsoriatic treatment (unchanged therapy in the preceding 3 months), while the other half of the patients do not receive systemic antipsoriatic treatment. The exclusion criteria are shown in
Dementia or other major psychological or physical incapacities
Other chronic systemic diseases
History of cancer with throat or thoracic irradiation or history of cancer with <3 years recurrence-free control, and for hematologic cancers, <5 years recurrence-free control
Major surgery, pregnancy, labor, or breastfeeding ≤6 months before inclusion
Immobility
Dysregulated diabetes (glycated hemoglobin >10%)
Systemic treatment with prednisolone or antibiotics <1 month before inclusion
Severe claustrophobia
Severe kidney disease (glomerular filtration rate <30 mL/min)
Inability to understand the information relating to participation in the study
Patients are recruited at the Department of Dermatology and Allergy, Herlev and Gentofte Hospital, when attending regularly scheduled visits for psoriasis control. Recruitment began in January 2018 and is also at the Department of Dermatology, Bispebjerg Hospital, at selected private dermatology clinics in the Copenhagen area, and through public advertisement of the research project on the home page of Herlev and Gentofte Hospital, social media outlets, and the member magazine of the Danish Psoriasis Association. All patient-related examinations are performed at Herlev and Gentofte Hospital. Because of the extensive examination program, the study visits are extended over 2 days of attendance within a period of 2 weeks.
Each participant completes a questionnaire containing questions regarding civil status, occupation, educational level, annual household income, psychological stress, depression, self-rated health, diet, and exercise. The questionnaire also contains questions to establish the Dermatology Quality of Life Index. An interview is conducted by study physicians with questions regarding history of psoriasis, current and previous medical treatment of psoriasis, history of CVD, medical treatment of CVD, family history of CVD, comorbidities (diabetes, hypertension, hypercholesterolemia, etc), odontological status, smoking status, alcohol consumption, other medications including use of antibiotics (exclusion if taken within 1 month before consideration for inclusion), and ethnicity.
A full skin examination is performed in each patient, and the severity of psoriasis is measured according to the Psoriasis Area and Severity Index and body surface area [
Isohelix DNA/RNA buccal swabs (SK-1S, Cell Projects Ltd) are used to collect samples from the skin. One swab is taken from a psoriasis plaque (if applicable), where the patient has at least 4 to 6 cm2 of affected skin. A second swab is taken from adjacent clinically healthy skin. The sample is taken by rubbing the skin with the swab for 60 seconds and is stored in DNA/RNA shield (Zymo Research Corp). Site-specific skin microbiomes vary between different body regions [
Two 4-mm skin punch biopsies are taken under local anesthesia, including one from active psoriatic skin and one from clinically healthy skin neighboring the psoriatic biopsy area with a minimum distance of 2 cm from the psoriasis biopsy. The biopsy locations have the same priority for body sites as the skin swabs. After collection, biopsies are immediately cut in two parts and processed separately for RNA sequencing and immunohistochemistry.
Biopsies for RNA sequencing are placed directly into RNAprotect Tubes (QIAGEN), which are stored at 4°C overnight and thereafter at −80°C. RNA is isolated from biopsies, and its quality is assessed using the 2100 Bioanalyzer (Agilent). Sequencing libraries are prepared using SureSelect XT RNA Direct (Illumina) for samples with an RNA integrity number (RIN) score >8 and are sequenced at the Genomics Core Facility at Icahn School of Medicine at Mount Sinai.
Biopsies for immunohistochemistry are placed into a cryomold (Tissue Tek, Sakura Finetek) with optimal cutting temperature (OCT) solution (Tissue Tek, Sakura Finetek) that is subsequently snap-frozen with liquid nitrogen and immediately stored at −80°C. OCT samples are cut, mounted, fixed, and stained with hematoxylin and eosin, and with antibodies to selected target antigens using standard immunohistochemical techniques.
Routine hematological and biochemical parameters, including high-sensitive C-reactive protein and N-terminal pro-brain natriuretic peptide, are analyzed at the Department of Clinical Biochemistry, Herlev and Gentofte Hospital, Denmark.
For mass cytometry profiling, blood is drawn in an ACD-A (acid citrate dextrose, Hettich Lab) tube, and 1 mL of blood is aliquoted into a tube containing 2 µL of Cell Activation Cocktail (phorbol 12-myristate-13-acetate [40.5 µM], ionomycin [669.3 µM], and Brefeldin A [2.5 mg/mL]; Biolegend). A second tube containing 1 mL of blood without the Cell Activation Cocktail is also collected. The tubes are mixed gently and then placed in an incubator at 37°C (5% CO2). After 6 hours, 1.4 mL of proteomic stabilizer (PROT1, Smart Tube Inc) is added to each tube and mixed. The tubes are incubated for 10 minutes at room temperature and are then immediately placed at −80°C. Immune cell populations and cytokines are analyzed via mass cytometry by time-of-flight (CyTOF) [
For profiling of plasma proteins, blood is collected in ethylenediaminetetraacetic acid (EDTA) tubes and centrifuged for 10 minutes at 2000 rpm. The plasma is aliquoted into 1-mL tubes and immediately stored at −80°C. Plasma concentrations of proteins are measured using the Olink Proseek multiplex assay (Olink Bioscience), which uses proximity extension assay technology to detect protein biomarkers in liquid samples [
Lipidomic and metabolomic profiling of plasma are assessed by Metabolon (Morrisville), where approximately 1100 lipids and 5200 metabolites are measured by ultra-high performance liquid chromatography/tandem mass spectrometry [
For transcriptomic profiling, blood (2.5 mL) is drawn into PAXgene RNA Tubes (BD Bioscience), placed at −20°C overnight, and then stored at −80°C. RNA is isolated using the PAXgene Blood RNA Kit (QIAGEN), and its quality is assessed using the 2100 Bioanalyzer (Agilent). Sequencing libraries are prepared using TruSeq Stranded Total RNA kits (Illumina) for samples with a RIN score >8 and are sequenced at the Genomics Core Facility at Icahn School of Medicine at Mount Sinai. Owing to the relatively small number of study subjects, genomics are currently not planned to be assessed in this work.
All patients receive an OMNIgene GUT kit (DNA Genotek) for collection of feces at a maximum of 14 days after the blood samples. The gut microbiome is analyzed by shotgun metagenomics [
A urine sample is collected, and the albumin-to-creatinine ratio is determined at the Department of Clinical Biochemistry, Herlev and Gentofte Hospital, Denmark.
Comprehensive two-dimensional resting transthoracic echocardiography, including tissue Doppler imaging, is performed using a Vivid E-95 ultrasound machine (GE Healthcare) with a M5Sc-D (1.4-4.6 MHz) transducer to determine myocardial structural and functional indices, such as left ventricular mass and systolic and diastolic function, right ventricular function, and left ventricular global longitudinal strain. Epicardial and pericardial adipose tissues are also measured in standard parasternal and short axis views [
At a maximum of 14 days after the blood samples are taken, all patients undergo 18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT). In brief, subjects are injected with 3.5 MBq (0.09 mCi) per kilogram FDG after fasting overnight. A whole-body FDG-PET/CT is performed 120 minutes after FDG injection on a GE Discovery 710 scanner (GE Medical Systems) using the proprietary Q.Clear PET reconstruction algorithm. Anatomic localization and attenuation correction are provided by an unenhanced low-dose CT scan. Regions of interest (ROIs) are delineated around the aorta in consecutive 3-mm-thick axial PET and CT slices using MIM 6.9.2 software (MIM Software Inc). FDG uptake in aortic segments is quantified by calculating the mean of the maximum activity in each ROI normalized to the mean activity in the superior vena cava (maximum target-to-background ratio [TBRmax]) to determine vascular inflammation according to established methodology [
Ultrasound imaging of CIMT is performed in the right and left common carotid arteries by using an Affiniti 70G ultrasound system with a 5-12 MHz linear array transducer (Philips Ultrasound Inc) with Philips Q-App IMT software (version 3.03). CIMT is measured according to the Mannheim consensus on a 10-mm far wall segment of the distal common carotid artery during diastole, avoiding areas with focal thickening [
For transcriptomics, RNA sequencing (RNA-seq) FASTQ sequence files are first subject to quality trim followed by alignment to the HG38 human genome and count summarization. Samples are normalized, and differential expression analysis is carried out to detect genes that are expressed at significantly different levels between groups. Gene set enrichment analysis is performed by the CAMERA method using reference data sets that include Hallmark, KEGG, Reactome, and Gene Ontology [
For metagenomics, FASTQ files are quality trimmed before human genome mapping to filter out human reads. Microbial taxonomy classification is performed by the k-mer–based Kraken2 tool supplied with a microbial database including archaea, bacteria, fungi, protozoa, viruses, and vectors [
For high dimensional CyTOF, data are first preprocessed in Cytobank, and then, FCS data files are imported into RStudio [
For proteomics, plasma protein levels are measured using the Cardiovascular II, Cardiovascular III, and Inflammation panels (Olink Bioscience), and reported as normalized protein expression levels (NPX) in log2 scale. Proteins are filtered out when 40% of samples are below the limit of detection. Protein changes are analyzed with the
The strength of a systems biology approach is the potential to integrate data from multiple platforms. There are several examples of how this approach has been successfully applied in the disease areas of psoriasis and in particular CVD [
The preliminary results of 18F-FDG-PET/CT data have recently been published, where vascular inflammation in patients treated with statins was significantly reduced in the ascending aorta (
The study has been approved by the ethics committee of the Capital Region, Denmark (H-17003458) and the local data protection agency (ID: HGH-2017-103, I-suite: 05977). All participants will be asked to sign an informed consent form before entering the study. The study has been conducted in agreement with the Declaration of Helsinki.
The ethics approval for the study was granted in March 2017. Recruitment for the study began in January 2018 and was completed in September 2020. Analyses of transcriptomics and proteomics have been completed, while analyses involving metagenomics, lipidomics, metabolomics, mass cytometry experiments, and immunohistochemistry are underway. Data processing and statistical analyses began in September 2020, and the first results of the study were published in the beginning of 2021 [
This is an observational study with a case-control design that includes adult patients with psoriasis with or without atherosclerotic cardiovascular disease, and with or without systemic antipsoriatic treatment. The major strength of this study is the extensive number of examinations and samples collected from each patient to achieve deep phenotypic characterization. High-throughput molecular profiling technologies and computational analyses are utilized, and data are integrated by multiscale network analyses. With this approach, results are likely to shed light on new drivers and mechanisms of cardiovascular risk in psoriasis that can impact precision medicine [
An important limitation of the study is that a control group without psoriasis is not included, which makes it impossible to compare the results with healthy individuals. Moreover, the computational analyses are data-driven and hypothesis-free, which precludes a priori sample size calculations. Another potential limitation is that patients with CVD are often older than those without CVD, so matching patients with and without CVD can be difficult. Furthermore, due to the extensive number of examinations and the 120 minutes of rest required for patients before the 18F-FDG-PET/CT scan, the scan is not performed on the day of clinical examination and collection of blood and skin samples, potentially leading to temporal changes in the state of systemic inflammation. All skin biopsies and swabs might not be taken from the same body regions in all patients due to variations in the location of psoriasis-affected skin. Moreover, because of the recruitment setup and study hospital localization, most patients will probably be recruited from the Copenhagen area, although patients from other parts of Denmark are eligible and can contact the project through social media or the member magazine of the Danish Psoriasis Association.
carotid artery intimamedia thickness
cardiovascular disease
mass cytometry by time-of-flight
fluorodeoxyglucose
interleukin
optimal cutting temperature
positron emission tomography/computed tomography
RNA integrity number
region of interest
T-helper
The study is supported by the LEO Foundation (grant LF16115). The LEO Foundation has no role in the design of the study; in the collection, analysis, and interpretation of data; and in writing this manuscript. Joel Dudley, PhD, and Brian Kidd, PhD, are acknowledged for their initial roles in the study design and the development of the molecular profiling strategy. We also acknowledge the Human Immune Monitoring Core and the Genomics Core Facility at Icahn School of Medicine at Mount Sinai for their contributions toward the study design and sample processing.
PRH and LS designed the study, and PRH is responsible for the study. HK and AKH are responsible for clinical recruitment, retrieval of biological samples, and overall data collection. MK, PMG, and KMAH performed the 18F-fluorodeoxyglucose positron emission tomography/computed tomography scan, carotid artery ultrasound, and echocardiography, respectively. CZ contributed to study oversight and recruitment. BDM is responsible for sample processing and designing the multiomic profiling protocols. CB and XW contributed toward the design of the multiomic profiling protocols and are responsible for sample processing and analysis of multiomic data. HK, AKH, LS, and PRH drafted the first manuscript. All authors have contributed to this revision of the manuscript, and have read and approved the final manuscript.
PRH is the recipient of a Borregaard clinical scientist fellowship from the Novo Nordisk Foundation and chairs a clinical academic group supported by the Greater Region of Copenhagen. CB is a consultant for Onegevity Health. LS has been a paid speaker for AbbVie, Eli Lilly, and LEO Pharma, and has been a consultant or has served on advisory boards for AbbVie, Janssen Cilag, Novartis, Eli Lilly, LEO Pharma, UCB, Admirall, and Sanofi. Moreover, she has served as an investigator for AbbVie, Janssen Cilag, Boehringer Ingelheim, AstraZeneca, Eli Lilly, Novartis, Regeneron, and LEO Pharma and has received research and educational grants from Pfizer, AbbVie, Novartis, Sanofi, Janssen Cilag, and LEO Pharma. CZ has been a scientific consultant, advisor, investigator, and speaker for Eli Lilly, Jansen Cilag, Novartis, Abb Vie, Takeda, Amgen, Almirall, CSL Behring, UCB, Regeneron, MSD, and LEO Pharma. HK, AKH, XW, BDM, MK, PMG, and KMAH declare that they have no competing interests.