Methods
Study Design and Population
The “Endocrine Vascular disease Approach” (EVA) project (
ClinicalTrials.gov identifier NCT02737982) was planned as an observational cohort prospective study. The flow chart of the study and the description of variables that will be considered are summarized in Table
1.
A registry will be built up of men and women aged 18 and older with suspected IHD who were referred to the catheterization laboratory in order to undergo coronary angiography and/or PCI. Patients will be recruited according to the eligibility criteria reported in Table
2. Therefore, the EVA study population will include patients with stable CAD, patients with non-ST elevation (NSTE) ACS, and patients with ST elevation myocardial infarction (STEMI).
Table 2EVA study population eligibility criteria
▪ Patients with ischemic heart disease (acute or chronic) undergoing percutaneous coronary intervention (urgent or elective) ▪ Written informed consent ▪ Both sexes ▪ Aged more than 18 years | ▪ Life expectancy less than 12 months ▪ Active cancer (i.e., chemotherapy or ≤ 5 years from diagnosis) ▪ Pregnancy ▪ Previous coronary artery bypass graft ▪ Documented moderate–severe valvular heart disease ▪ Biological or mechanical valves |
The study will be conducted in full conformance with the principles of the Declaration of Helsinki, the laws and regulations of Italy, or whichever affords the greater protection to the individual. The study has obtained the required authorization by the local Ethics Committee of Policlinico Umberto I, Sapienza University of Rome (reference 3786, 24/09/2015). Written informed consent will be obtained from all patients.
Among the study population, we will select (1) patients undergoing urgent PCI, specifically subjects who presented within 12 h of the onset of chest pain, who had ST segment elevation of more than 0.1 mV in two contiguous leads, and for whom the clinical decision was made to treat with PCI (STEMI); (2) patients with NSTE ACS defined by acute chest pain but no persistent ST segment elevation. ECG changes may include transient ST segment elevation, persistent or transient ST segment depression, T-wave inversion, flat T waves, or pseudo-normalization of T waves or the ECG may be normal. Myocardial ischemia can occur with (myocardial infarction–NSTEMI) or without cell loss (unstable angina); (3) patients undergoing coronary angiography and elective PCI for stable angina, with stress test inducible myocardial ischemia and de novo native coronary lesion with a stenosis of less 50% (no obstructive CAD) or > 50% stenosis in any epicardial artery (mono-vessel obstructive CAD). Patients will be treated according to standard guidelines based on the CAD type, either with PCI or coronary artery bypass graft.
The recruitment phase started in April 2016 and, currently, 200 patients have already been included in the study.
Clinical, Pharmacological, and Gender-Related Factors
We will record complete information regarding (1) anthropometric data including height, weight, blood pressure, and heart rate; (2) socioeconomic status and gender-related factors: marital status, education level, income, physical activity (self-administered questionnaire), independent functional status Duke Activity Status Index [
70], alcohol intake, smoking habits, perceived stress at work and at home, personality traits, and psychosocial characteristics and support [
12,
71]; (3) cardiovascular risk factors (including familiar history of cardiovascular disease; hypertension; dyslipidemia, diabetes, previous cardiovascular events including previous MI and prior stent implantation); (4) symptom evaluation through standardized questionnaires [
72,
73]; (5) dietary habits [
74]; and (6) pharmacological therapy and Morisky Medication Adherence Scale—4 Items [
75].
Coronary Angiography Assessment
Coronary angiography will be performed according to standard methods. Qualitative and quantitative coronary angiographic analyses will be conducted by a core laboratory blinded to patient data. Time of procedure and timing to access at the catheterization after the first medical contact will be collected to explore chronobiology aspects.
Laboratory Measurements
Routine blood tests (e.g., cardiac troponin, as myocardial injury index, whole blood count, creatinine level, glycemia, lipid profile, hepatic enzymes) performed during hospitalization will be recorded.
The collection of biological samples for studies of platelet function will be performed in accordance with the guidelines suggested by the international literature and at scheduled time points. Blood samples will be properly maintained until batch analysis in freezers at − 80 °C. All samples will be coded to ensure anonymity and entered in conjunction with clinical data in a computerized database protected by double access code/password. All assays will be performed in a blinded fashion. The samples analyzed by immunoassay methods will be tested in duplicate, and those with concentrations exceeding the standard curve will be assayed again after appropriate dilution. The results obtained will be compared with reference values obtained in our laboratory on a reference population (healthy subjects, both sexes, and aged from 25 to 80 years).
During the angiography, blood samples will be collected at coronary and peripheral levels before and after PCI. According to a previously reported study, the arterial sample will be suitable for testing biomarkers of platelet function [
77]. Moreover, a peripheral sample collection after 24 h will be performed.
Given the close interaction between thrombosis and hormone pathways, we plan to evaluate experimental biochemical markers of platelet function in association with sex hormone profiles.
In every patient, regardless of sex, we will perform the measurement of 17-beta estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and free testosterone (BioT). The BioT/E2 ratio will be calculated. For women, we will consider for the interpretation of results data concerning the menopausal status and, if in pre-menopause, the timing of the menstrual cycle in which the blood collection was performed.
To better understand sex differences, plasma, serum, and cell lysates opportunely prepared from the enrolled populations will be collected and managed in an appropriate manner. Furthermore, the platelet-rich plasma will be prepared for the study of platelet aggregation, and in vivo markers of platelet activation (i.e., thromboxane, soluble CD40 ligand, soluble P selectin, isoprostanes) will be detected. Apoptotic processes, protein expression of estrogens and androgens, and their signaling will all be measured in the cell lysates. Platelets will be used to study signal transduction pathways of sexual hormone receptors, nitric oxide synthase (NOS) phosphorylation, and oxidative stress pathways including NADPH oxidase 2 (NOX2). In these cell types, the protein expression of endocellular and membrane receptors will also be studied using Western blot. The involvement of the receptors will be investigated using specific inhibitors, hormones, and specific agonists.
Using flow cytometry, we will analyze platelet-derived microparticles and surface receptors (i.e., CD40 ligand, P-selectin, GPIIb/IIIa, CX3CR1, GPVI) from resting and activated platelets in vitro of patients with ACS versus age- and sex-matched healthy individuals. Therefore, we could evaluate the correlation between circulating molecules and membrane-associated markers.
CMR Sub-Study
With the aim of better describing the CMR pattern of CMD in IHD patients, we planned a pilot study including (1) patients (N = 10; ratio W/M = 1:1) with mono-vessel obstructive CAD undergoing PCI and (2) patients (N = 10; ratio W/M = 1:1) undergoing angiography that documented no obstructive coronary disease with an impairment of microvascular dysfunction defined during angiography by a MBG < 2. Exclusion criteria will be (1) standard contraindication to magnetic resonance; (2) severe arrhythmia or tachycardia; (3) history or evidence of systemic autoimmune disorders, sarcoidosis, amyloidosis, or congenital heart disease; and (4) chronic kidney disease stages 4 and 5 (glomerular filtration rate < 30 mL/min/1.73 m2).
The procedure will be performed within 7 days from angiography to detect coronary microvascular dysfunction related to the procedure according to standard protocol (i.e., first-pass perfusion and the delayed post-contrast sequences). All CMR studies will be analyzed off-line in consensus by two experienced observers blinded to clinical information, using a workstation with dedicated cardiac software.
Follow-Up Phase
The follow up-phase will include the in-hospital length of stay and 1 year after discharge. A phone call interview within 3 months and an ambulatory visit at 12 months will be performed.
Clinical, pharmacological, and behavioral information will be re-assessed at the time of follow-up. Information concerning the duration of in-hospital stay as well as any adverse events before discharge will be collected. The letter of discharge for each participant will be collected. Reassessment of medication adherence, as well as psychosocial and behavioral aspects will be checked at the scheduled follow-up as well as the occurrence of new comorbidities.
Adverse clinical outcomes will be determined through the medical chart reviews following the index angiography. Peri-procedural and after-discharge complications with focus on thrombotic and bleeding events will be recorded. Minor and major bleeding events according to International Society on Thrombosis and Hemostasis [
78] and Bleeding Academic Research Consortium [
79], as well as recurrence of angina symptoms will also be recorded during the follow-up phase.
All participants will be followed for 1 year to verify clinical endpoints: early (30 days) and late mortality (12 months) for cardiac-related events or for any other cause; recurrence of acute coronary syndrome; need for re-interventions or coronary artery bypass graft, complications related to PCI or re-admission for cardiac-related cause (i.e., heart failure), or for any other causes.
Data Collection, Management, and Quality Assurance
The EVA coordination group (V.R., M.P., S.B., A.L.) will have full access to data and they will be responsible for the data management, including quality check. Investigators will be responsible for data entry into the electronic case report forms (eCRFs). In case of discrepant data, the coordination group will request data clarification, and if this should not be sufficient, the patient will be excluded from the analysis. EVA collaborators will receive training and a have access to a checklist for appropriate eCRF completion. By using a validation plan integrated in the data entry software, data will be checked for missing or contradictory entries and values out of the normal range. A final database will be created and validated by the study coordinator. Patients’ identification names will be registered by recruiting personnel. A series of consecutive numbers will identify patients. Regular monthly meetings will be conducted to check the sustainability of the project.
Source documents (paper or electronic) in which patient data are recorded will be collected. They will include, but not be limited to, hospital records, clinical and office charts, laboratory notes, patient-reported outcomes, evaluation checklists, pharmacy dispensing records, copies of transcriptions that are certified after verification as being accurate and complete, patient files with radiological images, and records kept at medico-technical departments involved in the trial. Source documents, required to verify the validity and completeness of data, must be retained per the policy for retention of records.
Knowledge Translation
We will facilitate the dissemination of our research findings through publications (open access) and presentations at national and international congress meetings making scientific audiences aware of the findings and creating an interdisciplinary network that may influence both individual and public practice.
Statistical Analysis Plan
The data will be analyzed by appropriate statistics. Student’s t test and Pearson correlation will be used for normally distributed variables. When necessary, log transformation will be used to normalize the data, or appropriate non-parametric tests will be employed (Mann–Whitney U test and Spearman rank correlation test). Adjusted multivariate analyses will be used to assess the differences and relationships between angiographic, laboratory, and clinical characteristics, stratifying the analysis on sex- and gender-related variables. Among the pre-specified subgroup analyses, we will explore the clinical presentation of ischemia (stable vs. acute coronary syndrome), the type of disease (obstructive vs. non-obstructive disease), traditional cardiovascular risk factors, medication adherence, and the gender construct including gender identity, gender role, gender relations, and institutionalized gender. We will consider the interaction term between sex and hormones in the changes of platelet biomarkers and angiographic index of coronary flow.
All calculations will be made using appropriate statistical software packages.
Clinical Significance and Translational Outlook of the EVA Project
The application of sex and gender medicine is strongly recommended by the World Health Organization and other international organizations [
81‐
83]. The identification of biomarkers and therapeutic approaches that take sex and gender differences into proper consideration is crucial for the development of evidence-based medicine that reflects this important issue.
IHD is a complex and multifaceted syndrome and its pathophysiology includes different pieces of a single puzzle, including factors such as atherosclerosis, inflammation, platelet activation and vasospasm, as well as microcirculatory dysfunction worsened by abnormalities in endothelium, platelets, mitochondria, redox signaling, ion channels, and myocardium–blood flow cross-talk mismatch.
The EVA project will provide evidence regarding the contribution of platelet function and sex hormone balance in CMD. Moreover, the investigators expect to clarify the impact of biological sex and gender-sensitive variables on clinical outcomes after PCI procedure, including recurrence and re-hospitalization.
This study will inform on biological and psychosocial determinants of outcomes in IHD patients to be considered when developing a tailored approach aimed at improving the health of vulnerable patients. A better understanding of the interactions between sex hormones and platelet pathways involved in the development of ischemia will guide the development of potential pharmacological strategies based on sex, aiming, in turn, to modify the interaction between platelet and vascular responses in the setting of coronary angiography. Furthermore, the systematic collection of gender-related factors will contribute to the identification of the sex-specific impact of psycho-socio-cultural factors on clinical outcomes, providing new targets which will improve secondary prevention strategies and inform policies to foster the well-being of both women and men with IHD.
Acknowledgements
EVA collaborators: Claudio Tiberti, Federica Panimolle, Andrea Isidori, Elisa Giannetta, Laura Napoleone, Marta Novo, Silvia Quattrino, Simona Ceccarelli, Eleni Anastasiadou, Cinzia Marchese (Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy); Enrico Mangieri, Gaetano Tanzilli, Nicola Viceconte, Francesco Barillà, Carlo Gaudio, Evaristo Ettorre (Department of Cardiovascular, Respiratory, Nephrologic, Anaesthesiologic and Geriatric Sciences, Sapienza University of Rome, Rome, Italy); Giulio Francesco Romiti, Filippo Toriello, Eleonora Ruscio, Tommaso Todisco, Nicolò Sperduti, Giuseppe Santangelo, Giacomo Visioli, Marco Vano, Marco Borgi, Ludovica Maria Antonini, Silvia Robuffo, Claudia Tucci, Maria Virginia Savoia, Agostino Rossoni, Valeria Spugnardi, Annarita Vernile, Mariateresa Santoliquido, Verdiana Santori, Giulia Tosti, Fabrizio Recchia, Francesco Morricone, Roberto Scacciavillani, Alice Lipari, Andrea Zito, Floriana Testa, Giulia Ricci, Ilaria Vellucci, Marianna Vincenti, Silvia Pietropaolo, Daria Amoroso, Lucia Stefanini, Giovanni Talerico, Pasquale Pignatelli, Simona Bartimoccia, Roberto Cangemi, Salvatore Minisola, Sebastiano Filetti (Department of Internal Medicine and Medical Specialties, Sapienza University of Rome, Rome, Italy); Cristina Nocella (Department of AngioCardioNeurology, IRCCS NeuroMed, Pozzilli, Italy); Louise Pilote, Tabeth Tsitsi Jiri, Muhammad Ahmer Wali, Amanpreet Kaur (McGill University Health Centre Research Institute, Centre for Outcomes Research and Evaluation, Montreal, QC, Canada); Anna Rita Vestri, Adriana Servello (Department of Public Health and Infections Disease, Sapienza University of Rome, Rome, Italy); Patrizia Ferroni (San Raffaele Roma Open University and Inter-institutional Multidisciplinary Biobank, IRCCS San Raffaele Pisana, Rome, Italy); Clara Crescioli, Cristina Antinozzi, Francesca Serena Pignataro (Department of Movement, Human and Health Sciences Section of Health Sciences, Unit of Endocrinology, Università degli Studi di Roma “Foro Italico,” Rome, Italy); Tiziana Bellini, Alessandro Trentini (Department of Biomedical and Specialty Surgical Sciences, University of Ferrara, Ferrara, Italy); Roberto Carnevale (Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Latina, Italy); Carlo Catalano, Iacopo Carbone, Nicola Galea (Department of Radiological, Oncological and Pathological Sciences, Sapienza University of Rome, Rome, Italy); Giuliano Bertazzoni, Marianna Suppa, Antonello Rosa, Maria Gabriella Scarpellini (Department of Emergency Medicine, Policlinico Umberto I, Sapienza University of Rome, Rome, Italy); Alessandro Coppola (Chest Pain Unit, Policlinico Umberto I, Rome, Italy); Giulio Illuminati (The Department of Surgical Sciences, Sapienza University of Rome, Rome, Italy); Paola Mariani, Fabrizio Neri, Paolo Salis, Antonio Segatori, Laurent Tellini, Gianluca Costabile (Nursing Team Catheterization Lab Policlinico Umberto I, Rome, Italy).
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