Down-regulation of PTEN via miR-19b is associated with fibrosis in aortic stenosis in females
E Jover, K Ausin, A Fernandez-Celis, I Navarro-Salguero, B Otegui, A Vera, V Alvarez, R Sadaba, A Navarro, N Lopez-AndresAbstract
Background
Different mechanisms drive sex-dependent forms of aortic stenosis (AS), influencing the clinical presentation and therapeutic benefit in males and females. An extensive fibrotic remodelling is predominant in females with AS compared to the osteo-inflammatory phenotypes prevalent in males. Loss of PTEN, via mTOR activity, contributes to fibrosis in different pathological contexts. MiR-19b is up-regulated in the aortic valve (AV) and VICs of women with AS and targets PTEN.
Purpose
We sought to explore whether the higher basal expression of miR-19b in female VICs and AVs was associated with lower expression of PTEN and an enhanced fibrotic remodelling in females with AS.
Methods
The expression of miR-19b was studied in aortic valves (AVs) (whole AVs or sectioned into non-calcific, fibrotic or calcific areas) isolated from patients with severe AS undergoing elective surgical valve replacement (n=293, 60.6% men). Human valve interstitial cells (VICs, n≥3/sex) were also analysed. MiR-19b mimic and inhibition experiments were conducted in VICs from both sexes. The expression of PTEN, relevant fibrotic markers (e.g., COL1A1, COL3A1, FN, CTGF) or PI3K/Akt/mTOR signaling were analysed in clinical and in vitro samples. QPCR, ELISA and western blotting were used as analytical methods.
Results
Functional annotations revealed that miR19b potentially regulates ‘mTOR signaling pathway, ‘p53 signaling pathway’ and ‘Phosphatidylinositol signaling system’ all of which share PTEN as a common miR19b target. The expression of PTEN was lower in non-calcific and fibrotic areas of female AVs when compared to male AVs. Along the non-calcific-fibrotic-calcific areas, the expression of PTEN was inversely parallel to the expression of miR-19b in females (r= -0.335, p =0.036). In vitro studies on VICs confirmed a higher baseline expression of miR19b in female VICs and lower expression of PTEN compared to male VICs. Indeed, miR19b and PTEN were negatively correlated in female VICs. Exogenous up-regulation of miR-19b (miR-19b mimics) down-regulated PTEN and that was associated with enhanced pro-fibrotic markers, including collagen type I and type III, fibronectin and CTGF. Conversely, miR-19b inhibition led to PTEN up-regulation paralleled with a lowered expression of the pro-fibrotic markers. MiR-9b up-regulation was associated with an overactivation of the PI3K/Akt/mTOR pathway.
Conclusions
We reveal for the first time that miR19b/PTEN may prompt females to undergo trough predominant fibrotic phenotypes in AS. Our findings evidence a new mechanism by which miR19b might be responsible of sex-dependent phenotypes in AS paving new avenues for the development of mechanism-based therapies in female’s AS.