Hypoxic Culture Maintains Cell Growth of the Primary Human Valve Interstitial Cells with Stemness.

Kaho Kanno,Hironori Izutani,Shigeki Higashiyama,Junya Masumoto,Daisuke Nanba,Mika Hamaguchi,Tomohisa Sakaue,Mie Kurata,Kenji Namiguchi,Jun Aono

doi:10.3390/ijms221910534

Abstract

The characterization of aortic valve interstitial cells (VICs) cultured under optimal conditions is essential for understanding the molecular mechanisms underlying aortic valve stenosis. Here, we propose 2% hypoxia as an optimum VIC culture condition. Leaflets harvested from patients with aortic valve regurgitation were digested using collagenase and VICs were cultured under the 2% hypoxic condition. A significant increase in VIC growth was observed in 2% hypoxia (hypo-VICs), compared to normoxia (normo-VICs). RNA-sequencing revealed that downregulation of oxidative stress-marker genes (such as superoxide dismutase) and upregulation of cell cycle accelerators (such as cyclins) occurred in hypo-VICs. Accumulation of reactive oxygen species was observed in normo-VICs, indicating that low oxygen tension can avoid oxidative stress with cell-cycle arrest. Further mRNA quantifications revealed significant upregulation of several mesenchymal and hematopoietic progenitor markers, including CD34, in hypo-VICs. The stemness of hypo-VICs was confirmed using osteoblast differentiation assays, indicating that hypoxic culture is beneficial for maintaining growth and stemness, as well as for avoiding senescence via oxidative stress. The availability of hypoxic culture was also demonstrated in the molecular screening using proteomics. Therefore, hypoxic culture can be helpful for the identification of therapeutic targets and the evaluation of VIC molecular functions in vitro.

Highlights

Aortic valves are essential for maintaining the proper systemic hemodynamic by preventing backflow to the left ventricle
We have provided the first molecular evidence demonstrating the benefits of hypoxic culture for valve interstitial cells (VICs)
Sun et al reviewed the effects of exposing cells to oxidative stress, including senescence with proliferative arrest, CDKN1A upregulation, morphological changes, and senescence-associated, secretory phenotype related-cytokine production [15]

Summary

Introduction

Aortic valves are essential for maintaining the proper systemic hemodynamic by preventing backflow to the left ventricle. Stenotic valves with calcium depositions are often observed in people over the age of 65 years [1]. Valve leaflet are primarily comprised of endothelial cells, myofibroblasts, and valve interstitial cells (VICs). VICs regulate homeostasis of valve leaflet tissues by providing extracellular matrix that increases the valve’s resistance to mechanical stress. VICs contribute to valve calcification via their transformation to osteoblastic cells in the progression of aortic valve stenosis [2,3,4]. The expression of osteoblast-specific marker genes has been detected in calcified tissues in human samples. Only a few studies have investigated the biological characteristics of VIC-derived osteoblastic cell in vitro, and the signaling pathways underlying differentiation to osteoblasts remain poorly understood

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Conclusion