Nox2 and Nox4 influence neonatal c-kit⁺ cardiac precursor cell status and differentiation

Abstract

Redox status has emerged as critical in modulating stemness and lineage commitment in several precursor cell types. However, a role for redox genes, specifically NADPH oxidases (Nox), in cardiac precursor cells (CPCs) has not been established. We tested whether CPCs marked by type III receptor tyrosine kinase c-kit (c-kit(+)) exhibit a unique NADPH oxidase signature that confers precursor status and whether alterations in this profile are functionally linked to changes in lineage specification. Dihydroethidium (DHE) microfluorography indicated reduced basal reactive oxygen species (ROS) formation within early postnatal c-kit(+) CPCs. Real-time quantitative PCR revealed downregulation of ROS generator Nox2 and its subunit p67(phox) in c-kit(+) CPCs under basal conditions but upregulation of Nox2 and Nox4 over the course of differentiation. Adenoviral silencing of Nox2 and Nox4 increased expression of CPC markers c-kit and Flk-1 and blunted smooth and cardiac muscle differentiation, respectively, while overexpression of Nox2 and Nox4 significantly reduced c-kit expression. These changes were accompanied by altered expression of transcription factors regulating cardiac lineage commitment, Gata6 and Gata4, and cytokine transforming growth factor (TGF)-β1. Similar to other precursor cell types, RT(2)Profiler PCR Arrays revealed that c-kit(+) CPCs also exhibit enhanced antioxidant capacity at the mRNA level. In conclusion, we report that c-kit(+) CPCs demonstrate reduced Nox2 expression and ROS levels and that increases in Nox2 and Nox4 influence their differentiation into mature cells. We speculate that ROS generators Nox2 and Nox4, along with the antioxidant genes identified by PCR Arrays, may be novel targets in CPCs that could prove useful in cell-based therapy of the heart.

Nox2 and Nox4 regulate self-renewal of murine induced-pluripotent stem cells

“…For example, excessive amounts of ROS inhibited proliferation of iPSCs and MSCs , but the appropriate amount of ROS stimulated self‐renewal and proliferation of neural stem cells and adipose‐derived stem cells , which are consistent with our observation. On the other hand, Nox2‐ and Nox4‐derived ROS have been shown to play critical roles in cardiovascular cell differentiation . Our recent study also show that Nox2‐mediated ROS production contributes to the differentiation of miPSCs into endothelial cells (unpublished data), suggesting that the effect of ROS on cell proliferation and differentiation can differ profoundly, perhaps depending on other factors such as cell differentiation state, the amount of endogenous ROS, or other signaling co‐factors.…”

“…One of the primary cellular mechanisms of ROS production involves the oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) by NADPH oxidase (Nox) . Of the several Nox homologs expressed in stem cells, Nox2 and Nox4 are the most abundant ( ) , and the ROS produced by Nox2/Nox4 have been linked to many aspects of stem cell biology, including the proliferation of neural stem cells , stemness and proliferation in amniotic‐fluid stem cells , the mobilization of bone‐marrow progenitor cells in response to ischemic injury, chondrogenic , and neural‐stem cell differentiation , and the differentiation of cardiac precursor cells into smooth‐muscle cells and cardiomyocytes . Nox‐mediated ROS production also regulates migration and gene expression in stem cells , but the role of Nox2 and Nox4 in stem cell self‐renewal has yet to be thoroughly studied.…”

Regulation of Signal Transduction by Reactive Oxygen Species in the Cardiovascular System

“…Nadworny et al 77 induced the differentiation of c-kit+ cardiac precursor cells (CPCs) into mature cells and observed upregulation of Nox2 and Nox4 during differentiation. Furthermore, silencing of Nox2 and Nox4 increased expression of CPC genes such as c-kit and Flk-1, and decreased the expression of differentiation genes.…”

Peroxiredoxin-2 nitrosylation facilitates cardiomyogenesis of mouse embryonic stem cells via XBP-1s/PI3K pathway

“…Redox status has emerged as critical event in modulating stemness and lineage commitment towards cardiac precursor cell types. Among them, NADPH oxidases (Noxs) are essential components of cardiac redox biology and generate ROS in a highly regulated manner [47,48]. It has been well accepted that Nox activity enhances the cardiomyogenesis from ES cells via Nox-derived ROS pathways [22,49].…”