Exploring the regulation of circulating factors involved in aortic valve sclerosis onset

Abstract

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Ministero della Salute - Ricerca Finalizzata Background Aortic valve sclerosis (AVSc), the first stage of calcific aortic valve stenosis (CAVS), has a prevalence of 25-30% in subjects older than 65 years. Being a clinically silent phase, it is characterized only by non-uniform leaflet thickening due to fibrosis and microcalcifications, without the obstruction of the blood flow with an increased local oxidative stress, inflammation, and endothelial-to-mesenchymal transition (EndMT). However, to date, AVSc is not considered a pathology but 10% of cases evolve a full-blown CAVS. Aim Our aim is to investigate which systemic process(es) are activated in AVSc, in order to better define these subjects. These evaluations will allow us to identify potential druggable targets. Methods We consecutively enrolled 157 healthy subjects and detected AVSc presence by echocardiography. Plasma and serum samples were collected to evaluate circulating microRNA (RNA-sequencing) and cytokines (Luminex), respectively. Functional analysis was performed to reveal systemic pathway regulation. Healthy human valve endothelial cells were employed to evaluate viability (MTT assay) and EndMT (morphological analysis and quantitative PCR) Results AVSc was present in 77 patients (49%) and the differential expression analysis revealed that 59 microRNAs were down-regulated, while 93 were up-regulated in these subjects compared to healthy ones (adjusted-p<0.05). The functional analysis revealed that the major enriched pathways in AVSc subjects were related to inflammation. Indeed, serum GROα and IL1β levels were higher in AVSc vs healthy (p<0.05). In vitro, chronic GROα treatment resulted in lower cell viability (100.0±17.4 vs 61.8±16.5% of vital cells; p<0.0001), while IL1β treatment induced a decrease in cell roundness and an increase in total area (0.40±0.18 vs 0.47±0.19AU, p<0.0001; and 0.75±0.73 vs 0.45±0.48AU, p<0.0001; respectively). Quantitative PCR revealed EndMT activation: SNAI1 (1.87±0.57 log2(foldchange), p<0.0001) and SNAI2 (2.19±0.46 log2(foldchange), p<0.0001) only after IL1β treatment. Conclusions Inflammation is the most regulated systemic pathway in AVSc subjects and its master regulator, IL1β, seems to be involved in the EndMT progression of valve endothelial cells, while GROα contributes to their death. These effects could be related to the early triggers of AVSc onset, leading to fibrosis and eventually calcification of aortic leaflets.