Serum and Cellular Biomarkers for Aortic Valve Stenosis

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).