Serum Biomarkers for Aortic Valve Stenosis
Serum Biomarkers for Aortic Valve Stenosis
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 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.
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 and. 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.

 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) 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.

 (F) 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.

 (G) 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.

 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).
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