UNRAVELLING SEX-DEPENDENT MECHANISMS IN CALCIFIC AORTIC VALVE STENOSIS

Calcific aortic valve stenosis (CAVS) is the most common form of heart valve disease and affects about 3% of the population. Its prevalence increases with age, without a causal relation between ageing and CAVS development. To date, CAVS is a slow, progressive, multifactorial disorder considered to be actively driven by several cellular and molecular processes. Its natural history consists of a long clinically silent phase of non-uniform leaflet thickening with or without minimal calcification, known as aortic valve sclerosis (AVSc), without significant obstruction of blood flow, followed by the symptomatic stage, the aortic valve stenosis (AS). Currently, there is no pharmacological therapy preventing CAVS progression nor treating patients with AS. As a result, surgical or percutaneous aortic valve replacement remain the only treatments for severe AS, leaving the pathological molecular and cellular mechanisms unsolved. One of the first trigger of the pathology due to the oxidative stress is the endothelial dysfunction, followed by local inflammation and interstitial cells (VIC) differentiation into myofibroblasts and osteoblasts. Activated valve endothelial cells, undergoing endothelial to mesenchymal transition (EndMT), begin to express mesenchymal adhesion molecules and facilitate monocytes infiltration and local inflammation. These environmental changes induce VIC trans-differentiation into myofibroblast- and osteoblast-like cells. Activated VICs carry out a progressive extracellular matrix (ECM) pathological rearrangement characterized by the activation of fibrosis and calcification processes, which ultimately drive to fibro-calcific deposit formation. In the last years different studies reported sex-related difference in molecular mechanisms in the context of CAVS. In particular, it was shown that men with AS show a higher aortic valve calcium (AVC) load than women. Recently, it has been described that woman aortic valve leaflets were more fibrotic than man ones. Hence, it has been hypothesized that the mechanisms underlying CAVS progression could be different between the two sexes. We confirmed the evidence on sex-related calcium load in a meta-analysis performed on almost three thousand AS patients. Based on our results, AVC load, evaluated by computed tomography, is higher in man AS patients than in woman ones, even normalizing the data for the state of the pathology and for the aortic 9 annulus area. By the CT scan images analysis, we confirmed also the higher prevalence of fibrotic tissue in woman AS patients, than in men. In silico analysis of whole tissue RNA microarray revealed that the cellular composition of the aortic valve was different between men and women with CAVS. In particular, women showed a prevalence of mesenchymal cells, while in men there was a prevalence of inflammatory cells. This finding was in line with the analysis of circulating cytokines: pro inflammatory cytochines such as IL1β, TNFα, INFβ, and INFγ were upregulated in men CAVS patients. Based on these premises, we isolated and characterized VICs from AS patients and performed RNA sequencing to evaluate the differentially expressed molecular mechanisms. Among pathways overactivated in men there was the mitochondrial gene expression and this finding was confirmed by the higher mitochondrial damage in AS VICs from men respect to the one from women. We hypothesized that the mitochondrial damage caused a lower ATP production, therefore we evaluated the effects of a synthetic ATP equivalent, the 2ThioUTP, on the extracellular calcification of VICs from CAVS men. The in vitro 2ThioUTP administration showed indeed lower extracellular calcification of CAVS VICs both in normal and pro-calcifying conditions. All these data, taken together with robust literature evidences, shed light on the influence of sex in the development and progression of CAVS disease. Further studies are needed to better define the sexual dimorphism of this detrimental pathology. The recognition of sex-specific molecular mechanisms, linked to AS onset, may help in the identification of a gender-specific targeted therapy. In this direction, novel pharmacological therapies intended to reduce or even halt CAVS progression could be discovered, providing the basis for a personalized medicine approach in the context of CAVS.