Serum and Cellular Biomarkers for Aortic Valve Stenosis (AthenaValve)

Development of a Serum and Cellular Biomarkers Cluster for the Assessment of Degenerative Aortic Valve Stenosis Progression.

AthenaValve aims to develop and initial validate a novel serum diagnostic kit, for the assessment of severity and prognosis of progression of aortic valve stenosis (AS, a devastating disease without early diagnosis and medical treatment). Two independent clinical cohorts of patients will provide serum samples, along with tissue and serum of a validated animal model of the disease for evaluation of the early stages, in order to develop and validate a multiplexed Enzyme-linked Immunosorbent Assay kit (multiplex ELISA). Advanced bioinformatics analysis will facilitate the selection of the most promising molecules from integrated proteomics-transcriptomics-metabolomics data. The novel biomarkers will help clinicians to early diagnose patients at high risk and will pave the way for the experimental implementation of promising pharmaceutical therapies.

Moreover, AthenaValve aims to shed light on the systemic cellular interplay of the same patients, by analyzing the circulating immune cell phenotypes of the subgroups of rapid and slow progression patients

Study Overview

• Status: Recruiting
• Conditions: Aortic Valve Stenosis; Calcific Aortic Valve Disease; Aortic Valve, Calcification of
• Intervention / Treatment: Other: Untargeted LC/MS and LC-MS/MS; Diagnostic test: Echocardiography; Diagnostic test: Computerized Tomography Aortic Valve Calcium Score; Other: Targeted LC/MS and LC-MS/MS; Diagnostic test: multiplex ELISA

Detailed Description

The deposition of calcium salts in the arterial wall and the heart valves is a commonly occurring phenomenon associated with increasing age and various pathological factors. A particular case is the calcification of the aortic valve, which is accompanied by gradual stenosis leading to heart failure. As a result, causes significant morbidity and a dramatic increase in mortality if not treated surgically or via transcatheter intervention. It affects approximately 2% of the population over the age of 65, being the third cause of death in this population. It numbers about 100 surgeries per 100,000 person-years in the decade and 180000 patients each year are candidates for percutaneous aortic valve implantation (TAVI) in the US, Canada, and European Union.

Characteristic of the disease is that it remains even today without a diagnosis at the critical early stages and all existing therapies have failed to slow or reverse the damage when applied in the final stages. Previous experimental studies suggested that existing drugs may be more effective if given early in the disease course. At the time when the aortic stenosis becomes evident on echocardiography or Computerized Tomography (CT), underlying biochemical and molecular lesions have already developed. Moreover, among the aortic valve stenosis patients, it is unknown who will rapidly evolve to a dynamic narrowing of the valve and previous research studies report significantly diverse progression rates among patients, with approximately 20-25% demonstrating more than double rates. Thus, the Achilles heel of the therapeutic management of patients is the lack of early diagnosis and disease progression prognosis.

Under a combination of pathological effects by factors such as hypertension, diabetes mellitus and dyslipidemia in patients with a possible genetic predisposition, impairment of the normal structure and function of endothelium occurs, which progressively leads to the disease of the underlying valve tissue. Local acidosis and accumulation of oxidative oxygen radicals cause damage to membrane phospholipids, oxidation of cholesterol and proteins, which lose their normal quaternary structure and degrade. The gradual deposition of phosphate and calcium carbonate salts over the pathological proteins leads to calcification of the valve tissue. Intense inflammatory reaction with activation of bone marrow and infiltration of tissue with immunocompetent cells accompanies the damage: macrophages, B and T lymphocytes, mast cells infiltrate and actively contribute to the disease. Within this environment, inflammatory cytokines secreted from the inflammatory cells lead to activation of embryonic cell pathways that help to further enhance the calcification through the conversion of resident fibroblasts to active osteoblasts, which actively deposit calcium salts through extracellular vesicles.

In the disease, multiple cell types and distant organs are involved (liver - cholesterol metabolism, spleen and bone marrow - immune response activation) and the damage is expected to release fragments of biological and biochemical substances in the blood circulation. Hence, the disease is expected to be reflected in changes in serum protein. It is possible to detect proteins and metabolites in the blood serum in appropriately selected patients, prospectively enrolled and monitored through the course of aortic valve stenosis. An optimal subgroup description of patients with rapid or slow disease progression can reveal important information for future clinical diagnostic and prognostic use. Moreover, the disease is characterized by cellular infiltration of a plethora of known and unknown immune cells that contribute to the disease. Thus, the discovery of novel cellular phenotypes linked to disease progression can help delineate important clinical aspects.

The purposes of Athena Valve are:

(A) Collection of data on the progression of calcification and aortic valve stenosis, description of subpopulations with rapid and slow progression and record of clinical events.

(B) The bioinformatics analysis of data from combined transcriptomics-metabolomics-proteomics of diseased animal model aortic valve tissue in the early, intermediate and final stages, in comparison with existing patient multi-omics databases, in order to investigate candidate molecules - "drivers" of the damage with a causal relationship.

(C) The differential proteomic analysis of patient sera samples with severe aortic valve stenosis, retrospectively defined as rapid versus slow disease progressors and control group of individuals without aortic valve stenosis and severe calcification.

(D) The differential metabolomic analysis of patient sera samples with severe aortic valve stenosis, retrospectively defined as rapid versus slow disease progressors and control group of individuals without aortic valve stenosis and severe calcification.

(E) To obtain the differential profile of circulating immune cells among patients.

(F) The integrated bioinformatics data analysis of differential patient serum proteome and metabolome enriched with the results of the animal model and existing databases from other patients, to determine the most probable - causally related molecules with disease progression in a molecular network.

(G) Utilization of the more important protein targets for the development of a novel detection assay for multiple protein targets in blood serum by multiplex ELISA based on the results of the bioinformatics analysis.

(H) Application of the new assay multiplex ELISA in paired serum samples of patients with moderate to severe aortic stenosis and control group of individuals without aortic valve stenosis and severe calcification, prospectively enrolled and monitored, initial validation of the assay and review of its diagnostic performance.

Brief Methodology:

Two independent clinical cohorts will provide necessary data and serum samples. The first, a retrospective cohort of patients with severe aortic valve stenosis will define serum, echocardiographic and clinical differences among subgroups with different progression rates. The second cohort, prospectively developed, will confirm the retrospectively obtained clinical and echocardiographic data and provide baseline and follow-up measurements of aortic valve calcification (CT calcium score). Paired baseline and follow-up serum samples will validate serum targets from the retrospective cohort. Patients will be assessed with echocardiography annually and with follow-up CT at 2 and 5 years from enrollment.

Retrospective proteomics will be done with untargeted Liquid Chromatography/Mass Spectrometry (LC/MS) and metabolomics with untargeted Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) in Serial Reaction Monitoring (SRM) mode. Prospective proteomics and metabolomics will be quantitative and targeted. Blood samples will be immediately frozen and stored to -80 C.

Moreover, peripheral blood mononuclear cells will be isolated and cryopreserved at -80 C with differential density centrifugation. Cell populations will be analyzed utilizing mass cytometry, flow cytometry, cell sorting, and molecular methods.

For pathophysiological enrichment, a modified New Zealand rabbit model for aortic stenosis will be used in a longitudinal protocol of tissue harvesting in the early, intermediate and late-stage disease (corresponding to inflammation - calcification and subsequent valve stenosis).

• Study Type: Observational
• Enrollment (Anticipated): 280

Contacts and Locations

• Study Contact: Name: Nikolaos Anousakis-Vlachochristou, MD; Phone Number: 00302132088099; Email: anousakisvn@med.uoa.gr

Study Locations

• Greece
  • Athens, Greece
    • Recruiting
    • Naval Hospital of Athens
    • Contact: Nikolaos Anousakis-Vlachochristou, MD; Phone Number: 00302107261605; Email: anousakisvn@gmail.com
    • Principal Investigator: Nikolaos Anousakis-Vlachochristou, MD
  • Attiki
    • Athens, Attiki, Greece, 11528
      • Recruiting
      • First Department of Cardiology, University of Athens, Medical School. Hippocratio Hospital
      • Contact: Nikolaos Anousakis-Vlachochristou, MD; Phone Number: 00306936704929; Email: anousakisvn@med.uoa.gr
      • Contact: Nikolaos Ktenopoulos, MD; Phone Number: 00306977276366; Email: nikosktenop@gmail.com
      • Principal Investigator: Konstantinos Toutouzas, Professor

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 60 years to 87 years (Adult, Older Adult)
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: All

• Sampling Method: Probability Sample

Study Population

Patients diagnosed with moderate and severe tricuspid aortic valve stenosis of degenerative etiology.

Description

Inclusion Criteria:

• Retrospective cohort: patients with severe aortic valve stenosis in echocardiography:

  • Vmax > 4 m/sec and mean Gradient >40 mmHg and/or Aortic Valve Area indexed (AVAi) < 0.6 cm2/m2 and/or Velocity index <0.25 whichever worse, with available complete past echocardiographic follow-up (>2 past studies) indicating disease progression, by the same performing physician.

• Prospective cohort: patients with moderate aortic valve stenosis in echocardiography:

  • Vmax 3-4 m/sec and mean Gradient 25-40 mmHg and/or AVAi 0.6-0.9 cm2/m2,
  • Where inconsistent: Velocity index = 0.25-0.50.
  • Technical details: Optimal doppler measurements obtained by the best feasible echocardiographic window (demonstrating at least 2 windows, where possible including right parasternal with or without pencil probe).
• Prospective Control group: patients at intermediate to high risk for CVD according to atherosclerotic risk factors assessment - Heart Score

Exclusion Criteria:

• Echocardiographic Stroke Volume indexed (SVi) <35 ml/m2
• Bicuspid aortic valve
• Stenosis of rheumatic etiology
• More than mild aortic valve regurgitation
• More than mild mitral valve regurgitation
• More than mild mitral stenosis
• Severe pulmonary hypertension
• Chronic ischemic heart failure with Ejection Fraction < 45%
• Right heart failure (based on the echocardiographic assessment of Right Ventricle Dimension, Tricuspid Annular Plane Systolic Excursion, tricuspid annular velocity, and clinical syndrome)
• Acutely decompensated Heart Failure with preserved Ejection Fraction <4 weeks
• N-terminal-pro hormone Brain Natriuretic Peptide (NT-proBNP)> 900 pg/ml for ages 60-75, NT-pro-BNP > 1800 pg/ml for ages >75 years
• Presence of chronic systematic inflammatory disease
• Presence of autoimmune disease
• Active malignancy
• History of chemotherapy past 3 years
• Any history of thoracic radiotherapy
• Active treatment with monoclonal antibodies
• Under-treated hypertension
• Under-treated metabolic or endocrine disease
• Acute infection (<4 weeks)
• Any acute inflammation (<4 weeks)
• Unstable angina - myocardial infarction past 3 moths
• Stage 4 or 5 Chronic Kidney Disease (as defined by CKD-EPI derived estimated Glomerular Filtration Rate or 24hr urine measurement)
• Any acute renal failure <4 weeks
• Stroke past 3 months
• Any disabling stroke
• Surgery except for minor procedures past 3 months
• Poor mobility/immobilization
• Life expectancy < 3 years for any reason
• Difficulty in following follow-up protocol for any reason

For the control group:

• Known coronary artery disease
• Any heart valve disease, excluding minor valve regurgitation
• Any heart failure
• Pulmonary Hypertension
• Peripheral artery disease
• Presence of chronic systematic inflammatory disease
• Presence of autoimmune disease
• Active malignancy
• History of chemotherapy past 3 years
• Any history of thoracic radiotherapy
• Active treatment with monoclonal antibodies
• Under-treated hypertension
• Under-treated metabolic or endocrine disease
• Acute infection (<4 weeks)
• Any acute inflammation (<4 weeks)
• Stage 4 or 5 Chronic Kidney Disease, as defined by the Chronic Kidney Disease - Epidemiology Collaboration (CKD-EPI) derived estimated Glomerular Filtration Rate or 24hr urine measurement
• Any acute renal failure <4 weeks
• Any stroke
• Surgery except for minor procedures past 3 months
• Poor mobility/immobilization
• Life expectancy < 3 years for any reason
• Difficulty in following follow-up protocol for any reason

Study Plan

How is the study designed?

Design Details

• Observational Models: Case-Control
• Time Perspectives: Other

Number of groups / cohorts

4

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
Fast progressors of Aortic Valve Stenosis; 50 patients that retrospectively demonstrated fast progression from moderate to severe aortic valve stenosis: echocardiographic dVmax>0.25 m/sec/year (difference in transaortic Vmax).	Other: Untargeted LC/MS and LC-MS/MS; Serum samples untargeted proteomics and metabolomics
Slow progressors of Aortic Valve Stenosis; 50 patients that retrospectively demonstrated slow progression from moderate to severe aortic valve stenosis: echocardiographic dVmax<0.15 m/sec/year (difference in transaortic Vmax).	Other: Untargeted LC/MS and LC-MS/MS; Serum samples untargeted proteomics and metabolomics
Prospective Moderate Aortic Valve Stenosis; 100 consecutive prospectively enrolled patients with moderate aortic valve stenosis. Plus 20 patients accounting for 20% drop-out rate, total prospective cohort = 120 patients.	Diagnostic test: Echocardiography; Baseline and prospective follow-up echocardiography; Diagnostic test: Computerized Tomography Aortic Valve Calcium Score; Baseline and prospective follow-up CT calcium score of aortic valve; Other: Targeted LC/MS and LC-MS/MS; Serum samples targeted proteomics and metabolomics; Diagnostic test: multiplex ELISA; Implementation of novel multiplex ELISA assay on serum samples of aortic valve stenosis prospective cohort.
Negative calcium score group; 50 patients at intermediate to high risk for Cardiovascular Disease (CVD) and negative aortic valve and coronary calcium score at CT scan, prospectively followed-up. Plus 10 patients accounting for 20% drop-out rate, total control cohort = 60 patients.	Diagnostic test: Echocardiography; Baseline and prospective follow-up echocardiography; Diagnostic test: Computerized Tomography Aortic Valve Calcium Score; Baseline and prospective follow-up CT calcium score of aortic valve; Other: Targeted LC/MS and LC-MS/MS; Serum samples targeted proteomics and metabolomics; Diagnostic test: multiplex ELISA; Implementation of novel multiplex ELISA assay on serum samples of aortic valve stenosis prospective cohort.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
multiplex ELISA kit Sensitivity	Overall multiplex ELISA kit sensitivity for the detection of fast aortic valve stenosis progression (dVmax>0.25 m/sec/year)	5 years
multiplex ELISA kit Specificity	Overall multiplex ELISA kit specificity for the detection of fast aortic valve stenosis progression (dVmax>0.25 m/sec/year)	5 years

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Fast progressors percentage	Percentage of patients with echocardiographic aortic valve stenosis fast progression (dVmax>0.25 m/sec/year)	5 years
Severe AS percentage	Percentage of patients having echocardiographic Aortic Valve Area < 0.6 cm2/m2	5 years
Coronary Artery Disease progression percentage	Percentage of angiographically significant stable Coronary Artery Disease (CAD) treated with percutaneous coronary intervention or coronary artery bypass grafting, in patients without previous CAD.	5 years
Major Adverse Cardiovascular Events rate	Composite Major Adverse Cardiovascular Events (MACE), percentage of patients with cardiac death or non-fatal myocardial infarction or non-fatal stroke.	5 years

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Differential PBMC profile	ROC curve of circulating cell subpopulations discriminating patients from healthy volunteers and fast from slow progressors	5 years

Collaborators and Investigators

• Sponsor: National and Kapodistrian University of Athens
• Collaborators: University of Oxford; Naval Hospital of Athens; Biomedical Research Foundation of the Academy of Athens
• Investigators: Principal Investigator: Konstantinos P Toutouzas, Professor, First Department of Cardiology, Athens Medical School, NKUA

Study record dates

Study Major Dates

• Study Start (Actual): March 1, 2020
• Primary Completion (Anticipated): March 1, 2025
• Study Completion (Anticipated): September 1, 2025

Study Registration Dates

• First Submitted: March 14, 2020
• First Submitted That Met QC Criteria: March 14, 2020
• First Posted (Actual): March 18, 2020

Study Record Updates

• Last Update Posted (Actual): October 4, 2022
• Last Update Submitted That Met QC Criteria: October 1, 2022
• Last Verified: October 1, 2022

More Information

Terms related to this study

• Keywords: aortic stenosis; aortic valve stenosis; calcific aortic valve disease; biomineralization; calcification; cardiovascular calcification; serum diagnostics; multiplex ELISA; LC-MS/MS; bioinformatics; disease progression; Integrated proteomics-metabolomics-transcriptomics; Mass cytometry; Flow cytometry; Immune cells
• Additional Relevant MeSH Terms: Heart Diseases; Cardiovascular Diseases; Metabolic Diseases; Pathological Conditions, Anatomical; Heart Valve Diseases; Ventricular Outflow Obstruction; Calcium Metabolism Disorders; Aortic Valve Stenosis; Constriction, Pathologic; Aortic Valve Disease; Calcinosis; Physiological Effects of Drugs; Molecular Mechanisms of Pharmacological Action; Anti-Infective Agents; Enzyme Inhibitors; Anti-Bacterial Agents; Protein Synthesis Inhibitors; Calcium-Regulating Hormones and Agents; Calcium; Lincomycin
• Other Study ID Numbers: Τ2ΕΔΚ-01683

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: No