Over-expression of neurotrophin 3 in human aortic valves affected by calcific disease induces the osteogenic responses via the Trk-Akt pathway

Abstract

Calcific aortic valve disease (CAVD) is a leading cardiovascular disorder in the elderly. While aortic valve interstitial cells (AVICs) are the main cells that express osteogenic mediators, the molecular mechanism that mediates AVIC osteogenic responses is incompletely understood. This study aims to identify pro-osteogenic factors in human AVICs affected by CAVD.

Methods and results: Microarray analysis identified 11 up-regulated genes in AVICs of diseased valves. Among these genes, mRNA levels of neurotrophin 3 (NT3) increased by 2 fold. Higher levels of NT3 protein in diseased aortic valves and diseased AVICs were confirmed by immunofluorescent staining and immunoblotting, respectively. An exposure of AVICs of normal valves to recombinant human NT3 (0.025-0.10μg/mL) up-regulated the production of Runx2, TGF-β1 and BMP-2 in a dose-dependent fashion. NT3 also promotes calcium deposit formation. The pro-osteogenic effect of NT3 was not affected by neutralization of Toll-like receptor 2 or 4. Interestingly, mRNA encoding neural growth factor receptors (TrkA, TrkB, TrkC and p75 NTR) was detectable in human AVICs. Inhibition of Trk receptors markedly reduced the effects of NT3 on Runx2, TGF-β1 and BMP-2 production, calcium deposit formation and Akt phosphorylation. Further, inhibition of Akt also reduced the pro-osteogenic effects of NT3.

Conclusions: AVICs of diseased human aortic valves express higher levels of NT3. NT3 up-regulates the production of Runx2, TGF-β1 and BMP-2, and promotes calcium deposit formation in human AVICs via the Trk-Akt pathway. Thus, NT3 is a novel pro-osteogenic factor in aortic valves and may play a role in valvular calcification.

Keywords: Akt; Aortic valve; Neurotrophin 3; Pro-osteogenic mediators; Trk.