New markers for cardiovascular disorders

The main goal of the present application is to discover and establish new molecular markers that allow to assess the risk for cardiovascular disease (CVD) in human beings. Based on recent evidence that the accumulation of senescent endothelial cells is a key event in the development of cardiovascular disease, we will use soluble proteins secreted by senescent endothelial cells as potential markers for early detection of the disease. In a first step, we will try to identify suitable candidate genes that could give rise to such extracellular marker proteins, using a cell culture model in combination with the analysis of healthy biopsies from donors of various age groups. In a second step, clinical material will be used to validate the findings and lay the ground for the development of a new diagnostic kit. To achieve these objectives, the following experiments will be carried out: First, we will identify secreted and membrane-associated proteins that are overexpressed in senescent endothelial cells (HUVEC) and released to the extracellular space. This will be followed by the generation of antisera for selected candidate proteins, which will allow to study abundance of the proteins in HUVEC extracts and supernatants. Further identification of candidate genes will rely on RNA in situ hybridization analysis to establish changes in gene expression occurring in blood vessels from elderly donors and in diseased tissues. For clinical validation of our findings, we will also attempt to develop ELISA assays for selected gene products and test them in sera derived from CVD patients and healthy controls. This will be complemented by a feasibility study, where we will try to develop cytobead-derived test systems, in collaboration with a company.

The main goal of the project was the identification of new protein markers for early detection of cardiovascular diseases. The project hypothesis is that senescent human endothelial cells play an important etiological role in the pathology of cardiovascular diseases. The first goal of the project was the identification of potential marker proteins, which are upregulated in sera of people with subclinical endothelial dysfunction. According to the literature, senescent cells play a positive functional role in cardiovascular diseases. We have identified in a systematic screening using proteomic and transcriptomic analysis about forty extracellular proteins, which are increasingly expressed and secreted by senescent endothelial cells, which should be detectable in patient sera. To discriminate functional from correlative biomarkers for selected secreted proteins, functional analysis was carried out. This revealed that three proteins, IGFBP-3, TL1A and IGFBP-6, are not only upregulated in supernatants of senescent cells, but play an important role as regulators of senescence. In an additional project part selected genes of the group mentioned above were analyzed with non-radioactive RNA in situ hybridization. We looked for genes, the expression of which is upregulated in vascular biopsies from old donors as well as young donors with cardiovascular disease. This study will be finished soon. In addition, we analyzed the expression of a few non- secreted proteins in the same biopsies, which led to quite interesting results in particular for the CDK inhibitor p16. In collaboration with the group of Giovanni Grillari (BOKU University of Vienna), monoclonal antibodies for two selected secreted proteins were produced. The establishment and testing of sandwich ELISAs based on these antibodies will be finished in about 1-2 years. In summary, this project created the basis for the development of a diagnostic kit, which once finished will be tested in clinical pilot studies for its suitability to detect subclinical disease in apparently healthy donors.