Abstract
Introduction: Endothelial progenitor cells (EPCs) display a unique ability to promote angiogenesis and restore endothelial function in injured blood vessels. NADPH oxidase 4 (NOX4)-derived hydrogen peroxide (H2O2) serves as a signaling molecule and promotes endothelial cell proliferation and migration as well as protecting against cell death. However, the role of NOX4 in EPC function is not completely understood.Methods: EPCs were isolated from human saphenous vein and mammary artery discarded during bypass surgery. NOX4 gene and protein expression in EPCs were measured by real time-PCR and Western blot analysis respectively. NOX4 gene expression was inhibited using an adenoviral vector expressing human NOX4 shRNA (Ad-NOX4i). H2O2 production was measured by Amplex red assay. EPC migration was evaluated using a transwell migration assay. EPC proliferation and viability were measured using trypan blue counts.Results: Inhibition of NOX4 using Ad-NOX4i reduced Nox4 gene and protein expression as well as H2O2 formation in EPCs. Inhibition of NOX4-derived H2O2 decreased both proliferation and migration of EPCs. Interestingly, pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) decreased NOX4 expression and reduced survival of EPCs. However, the survival of EPCs was further diminished by TNF-α in NOX4-knockdown cells, suggesting that NOX4 has a protective role in EPCs.Conclusion: These findings suggest that NOX4-type NADPH oxidase is important for proliferation and migration functions of EPCs and protects against pro-inflammatory cytokine induced EPC death. These properties of NOX4 may facilitate the efficient function of EPCs which is vital for successful neovascularization.
Highlights
Endothelial progenitor cells (EPCs) display a unique ability to promote angiogenesis and restore endothelial function in injured blood vessels
Currently there is no defining characteristic of EPCs, these cells are primarily identified by expression of haematopoietic stem cell surface antigens CD133, CD34, and Vascular endothelial growth factor receptor 2 (VEGFR2) (Yin et al, 1997; Peichev et al, 2000; Urbich and Dimmeler, 2004; Friedrich et al, 2006; Zengin et al, 2006; Chang et al, 2007; Chen et al, 2007; Ranjan et al, 2009)
EPCs were isolated from the luminal wall of human mammary artery/saphenous vein and expanded in vitro under low cell density culture conditions (103 cells/cm2)
Summary
Endothelial progenitor cells (EPCs) display a unique ability to promote angiogenesis and restore endothelial function in injured blood vessels. Evidence continues to accumulate regarding the importance of EPCs for restoring endothelial function in injured blood vessels and neovascularization in ischemic tissues (Asahara et al, 1999a; Assmus et al, 2002; Iba et al, 2002; Griese et al, 2003; Krankel et al, 2012; Galasso et al, 2013; Lee et al, 2016). Early EPCs do not proliferate in vitro (Rehman et al, 2003) whereas, late EPCs are highly proliferative, differentiate into mature endothelial cells and are directly incorporated into blood vessels during neovascularization (Hur et al, 2004; Ranjan et al, 2009; Cheng et al, 2013)
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