NADPH oxidase (Nox4) produces reactive oxygen species (ROS) that are important for vascular smooth muscle cell (SMC) behavior, but the potential impact of Nox4 in stem cell differentiation is unknown. When mouse embryonic stem (ES) cells were plated on collagen IV-coated dishes/flasks, a panel of SMC-specific genes was significantly and consistently upregulated. Nox4 expression was markedly correlated with such a gene induction as confirmed by real-time PCR, immunofluorescence, and Western blot analysis. Overexpression of Nox4 specifically resulted in increased SMC marker production, whereas knockdown of Nox4 induced a decrease. Furthermore, SMC-specific transcription factors, including serum response factor (SRF) and myocardin were activated by Nox4 gene expression. Moreover, Nox4 was demonstrated to drive SMC differentiation through generation of H(2)O(2). Confocal microscopy analysis indicates that SRF was translocated into the nucleus during SMC differentiation in which SRF was phosphorylated. Additionally, autosecreted transforming growth factor (TGF)-beta(1) activated Nox4 and promoted SMC differentiation. Interestingly, cell lines generated from stem cells by Nox4 transfection and G418 selection displayed a characteristic of mature SMCs, including expression of SMC markers and cells with contractile function. Thus we demonstrate for the first time that Nox4 is crucial for SMC differentiation from ES cells, and enforced Nox4 expression can maintain differentiation status and functional features of stem cell-derived SMCs, highlighting its impact on vessel formation in vivo and vascular tissue engineering in the future.

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To investigate the role of miRNA in controlling human embryonic stem (hES) cell differentiation toward the endothelial lineage and chick embryonic blood vessel formation, undifferentiated hES cells were first cultured on Matrigelcoated flasks and in endothelial cell growth medium-2 (EGM-2) to initiate endothelial cell (EC) differentiation.

Objective-Phospholipase A 2 , group 7 (Pla2g7) is an important mediator in cardiovascular development and diseases because of its divergent physiological and pathological functions in inflammation and oxidative stress. However, little is known about the functional role of Pla2g7 in smooth muscle cell (SMC) differentiation from stem cells. Methods and Results-In the present study, embryonic stem cells were cultivated on collagen IV-coated plates to allow SMC differentiation. Pla2g7 gene expression and activity were upregulated significantly following 4 to 14 days of cell differentiation and colocalized with SMC differentiation markers in the differentiated SMCs. Knockdown of Pla2g7 resulted in downregulation of smooth muscle-specific markers in vitro and impairment of SMC differentiation in vivo, whereas enforced expression of Pla2g7 enhanced SMC differentiation and increased reactive oxygen species generation. Importantly, enforced expression of Pla2g7 significantly increased the binding of serum response factor to SMC differentiation gene promoters, resulting in SMC differentiation, which was abolished by free radical scavenger and flavoprotein inhibitor of NADPH oxidase but not hydrogen peroxide inhibitor. Moreover, we demonstrated that nuclear factor erythroid 2-related factor 3 (Nrf3) 8 -9 ; this differentiation is essential for vascular tissue engineering. Moreover, growing evidence suggests that stem/ progenitor cells can differentiate into vascular endothelial cells to repair damaged vascular tissues 8 -9 and SMCs to participate in neointimal formation and development 10 -12 in vivo. Therefore, it is important to understand the molecular pathways and specific gene program governing the distinct differentiation of stem cell toward vascular SMCs that could provide important insights into molecular mechanisms of SMC differentiation and novel therapeutic targets for prevention or intervention of cardiovascular diseases. To identify such signaling pathways involved in SMC differentiation, we have successfully established an efficient in vitro model for ES cell differentiation into SMCs. 5 Importantly, by using this model, we demonstrated that reactive oxygen species (ROS) generation and balance are essential for SMC differentiation from stem cells and successfully identified NADPH oxidase 4 (Nox4) 3 and the transcription factor nuclear factor erythroid 2-related factor 3 (Nrf3) 2 as 2 important regulators of ROS balance and SMC differentiation. However, the detailed molecular mechanisms of SMC differentiation from stem cells have not been fully clarified.The plasma isoform of a platelet-activating factor (PAF) acetylhydrolase, also known as lipoprotein associated phospholipase A 2 , encoded by the phospholipase A 2 , group 7 (Pla2g7) gene in humans, is an enzyme that catalyze hydrolysis of the sn-2 ester bond of PAF and related proinflammatory phospholipids. To maintain consistency, we refer both PAF acetylhydrolase and Lp-Pla2 as Pla2g7 in this text. A substantial body of peer-reviewed population studies h...