Oxidative‐mechanical stress signals stem cell niche mediated Lrp5 osteogenesis in eNOS^−/− null mice

Abstract

Introduction Calcific aortic valve disease(CAVD) is the most common indication for valve surgery in the USA. This study hypothesizes that calcific aortic valve disease develops secondary to Wnt3a/Lrp5 activation via oxidative-mechanical stress in eNOS null mice. Methods eNOS−/− mice were tested with experimental diets including a control (n=20), cholesterol (n=20), cholesterol + Atorvastatin (n=20). After 23 weeks the mice were tested for the development of aortic stenosis by Echo, Histology, MicroCT, and RTPCR for bone markers. In vitro studies measured Wnt3a secretion from aortic valve endothelial cells and confirmed oxidative stress via eNOS activity. Anion exchange chromatography was performed to isolate the mitogenic protein. Myofibroblast cells were tested to induce bone formation. Results Cholesterol treated eNOS mice develop severe stenosis with an increase in Wnt3a, Lrp5, Cbfa1, (3-fold increase(p<0.0001) in the bicuspid versus tricuspid aortic valves. Secretion of Wnt3a from aortic valve endothelium in the presence of abnormal oxidative stress was correlated with diminished eNOS enzymatic activity and tissue nitrite levels. Initial characterization of the architecture for a stem cell nice was determined by protein isolation using Anion-Exchange Chromatography and cell proliferation via thymidine incorporation. Osteoblastogenesis in the myofibroblast cell occurred via Lrp5 receptor upregulation in the presence of osteogenic media. Conclusion Targeting the Wnt3a/Lrp5 pathway in valve calcification and activation of osteogenesis is via an oxidative-mechanical stress in CAVD. These findings provide a foundation for treating this disease process by targeting the cross talk mechanism in a resident stem cell niche.

The pathology and pathobiology of bicuspid aortic valve: State of the art and novel research perspectives

“…Studies indicate that nitric oxide (NO) could modulate mineralisation and lower the expression of osteoblastic genes in vascular cells. In this regard, eNOS ‐/‐ mice under a cholesterol‐rich diet develop CAVD and mice with BAV have higher levels of Wnt3a, Lrp5 and Runx2 65. These data suggest that eNOS‐derived nitric oxide modulates the Wnt/Lrp5 pathway, which has been found to promote mineralisation of the aortic valve in patients with CAVD 18.…”

Early Development of Calcific Aortic Valve Disease and Left Ventricular Hypertrophy in a Mouse Model of Combined Dyslipidemia and Type 2 Diabetes Mellitus

“…[3][4][5] In this regard, various pathogenic processes such as lipid infiltration/retention, inflammation, and osteogenesis are now considered as key drivers of aortic valve mineralization. [6][7][8] A dysmetabolic state, characterized by insulin resistance/T2D, has been shown to play an important role in the development of aortic valve mineralization and the progression of AS.…”

Recent Advances in Research on Human Aortic Valve Calcification

“…Alfieri et al (57) reported that in interstitial cells exposed to culture medium for ossification, Wnt3a provoked nuclear translocation of b-catenin and caused a change in the expression of ossification-related genes. Furthermore, oxidative stress causes AVICs to transform into osteoblasts via the Wnt3a/Lrg5 pathway (58). This demonstrates that downstream signaling may differ according to the type of stimulus for calcification and that we can expect drugs targeting calcification-related signaling to have various effects.…”