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Extracellular vesicles as intercellular messengers in aortic valve calcification

Although aortic valve stenosis (AS) represents the most common type of valvular disease, its pathogenesis remains poorly understood. Recent evidence of the mechanisms of vascular disease and calcification-related processes indicates that extracellular vesicles (EVs) might play an important role in these processes. EVs are important mediators in intercellular communication and contain specific molecular cargoes, including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) that can modify specific cellular targets. Moreover, since vesicle composition and content reflect specific phenotypes/conditions of the releasing cells, EVs represent useful tools for diagnosing and monitoring cardiovascular as well as other diseases. However, the regulation of EV content and release and the mechanisms of interaction between EVs and the target cells involved in AS remain unclear. As there is accumulating evidence for the impact of EVs on endothelial cells and the vascular wall, EVs and their cargo might influence also the development of aortic aneurysms (AA).

We hypothesize that EVs and EV-associated ncRNAs are actively involved in the regulation of aortic disease consisting of aortic valve stenosis and aortic aneurysms.

The initial goal of the proposed project is to explore the biological content and function of EVs under differentconditions (calcifying vs. non-calcifying) and their contribution to valvular calcification processes. Both microvesicles and exosomes will be studied, with a focus on the miRNAs delivered by these EVs. In addition, also other noncoding (nc) RNA species will be analyzed. In Aim 1, we will analyze miRNA/lncRNA content in EVs isolated from AS patient samples. For this, we will collect blood and aortic valve tissue from diseased patients (Bonn Aortic Valve Tissue and Blood Bank). Hereby, we will profile EV-derived ncRNAs in these patients and find potential ncRNA candidates that will be analyzed in further detail for their pathophysiological function as well as their potential use as a biomarker for AS. In our preliminary experiments, we found that various miRNAs (miRNA-122, miRNA-92a) and lncRNAs (AGAP2-AS1, MALAT1) expressed in the aortic valve of diseased patients are differentially regulated compared to the control group. Aim 2 will focus on the contribution of EVs to valvular calcification in in vitro models. We will run target prediction analysis on EV-derived miRNAs and lncRNAs from Aim 1 and then test promising candidates in vitro to study their role and function during calcification. Aim 3 will focus on the role of pro-calcific vs. non-calcific EV-derived ncRNA in vivo. Specifically, we will test the development of AS following the gain- or loss-of-function of identified miRNA/lncRNA candidates in animal models. Additionally, we will investigate the contribution of EV-derived ncRNA on the development of AA within Aim 4 using a similar mechanistical approach as described for AS.

Our long-term objective is to elucidate ncRNA-controlled pathways during AS, which can be directly targeted using small molecules or RNA-based therapeutics. The detailed understanding of EV biology in AS and AA could also be used to directly deliver novel therapies to the location of disease, with the help of selectively loaded and specifically targeted EVs. Moreover, another long-term goal of this study is to implement valid ncRNA-based biomarkers for the occurrence and progression of aortic disease.




B04 Hypothesis: Intercellular communication between donor and target cells (VECs and VICs) via extracellular vesicles. The aortic valve consists of two main cell populations: valvular endothelial cells (VECs) and valvular interstitial cells (VICs). Upon stimulus (e.g. shear stress), VECs and VICs may release extracellular vesicles (EVs) with pro-calcific features. Pro-calcifying EVs transport proteins and miRNAs, which are able to modify target cells and trigger osteogenic differentiation, which is critical to calcification processes and AS. Furthermore, EV-contained ncRNAs may play a role as a biomarker for AS.

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