Crucial role of nrf3 in smooth muscle cell differentiation from stem cells.

RATIONALE: Nuclear factor erythroid 2-related factor (Nrf)3, a member of the cap 'N' collar family of transcription factors that bind to the DNA-antioxidant responsive elements, is involved in reactive oxygen species balancing and in muscle precursor migration during early embryo development. OBJECTIVE: To investigate the functional role of Nrf3 in smooth muscle cell (SMC) differentiation in vitro and in vivo. METHODS AND RESULTS: Nrf3 was upregulated significantly following 1 to 8 days of SMC differentiation. Knockdown of Nrf3 resulted in downregulation of smooth muscle specific markers expression, whereas enforced expression of Nrf3 enhanced SMC differentiation in a dose-dependent manner. SMC-specific transcription factor myocardin, but not serum response factor, was significantly upregulated by Nrf3 overexpression. Strikingly, the binding of SRF and myocardin to the promoter of smooth muscle differentiation genes was dramatically increased by Nrf3 overexpression, and Nrf3 can directly bind to the promoters of SMC differentiation genes as demonstrated by chromatin immunoprecipitation assay. Moreover, NADPH-derived reactive oxygen species production during SMC differentiation was further enhanced by Nrf3 overexpression through upregulation of NADPH oxidase and inhibition of antioxidant signaling pathway. In addition, Nrf3 was involved in the endoplasmic reticulum stressor induced SMC differentiation. CONCLUSION: Our findings demonstrate for the first time that Nrf3 has a crucial role in SMC differentiation from stem cells indicating that Nrf3 could be a potential target for manipulation of stem cell differentiation toward vascular lineage.

Histone deacetylase 3 is critical in endothelial survival and atherosclerosis development in response to disturbed flow.

BACKGROUND: Histone deacetylase 3 (HDAC3) is known to play a crucial role in the differentiation of endothelial progenitors. The role of HDAC3 in mature endothelial cells, however, is not well understood. Here, we investigated the function of HDAC3 in preserving endothelial integrity in areas of disturbed blood flow, ie, bifurcation areas prone to atherosclerosis development. METHODS AND RESULTS: En face staining of aortas from apolipoprotein E-knockout mice revealed increased expression of HDAC3, specifically in these branching areas in vivo, whereas rapid upregulation of HDAC3 protein was observed in endothelial cells exposed to disturbed flow in vitro. Interestingly, phosphorylation of HDAC3 at serine/threonine was observed in these cells, suggesting that disturbed flow leads to posttranscriptional modification and stabilization of the HDAC3 protein. Coimmunoprecipitation experiments showed that HDAC3 and Akt form a complex. Using a series of constructs harboring deletions, we found residues 136 to 206 of HDAC3 to be crucial in this interaction. Enforced expression of HDAC3 resulted in increased phosphorylation of Akt and upregulation of its kinase activity. In line with these findings, knockdown of HDAC3 with lentiviral vectors (shHDAC3) led to a dramatic decrease in cell survival accompanied by apoptosis in endothelial cells. In aortic isografts of apolipoprotein E-knockout mice treated with shHDAC3, a robust atherosclerotic lesion was formed. Surprisingly, 3 of the 8 mice that received shHDAC3-infected grafts died within 2 days after the operation. Miller staining of the isografts revealed disruption of the basement membrane and rupture of the vessel. CONCLUSIONS: Our findings demonstrated that HDAC3 serves as an essential prosurvival molecule with a critical role in maintaining the endothelial integrity via Akt activation and that severe atherosclerosis and vessel rupture in isografted vessels of apolipoprotein E-knockout mice occur when HDAC3 is knocked down.

Sustained activation of XBP1 splicing leads to endothelial apoptosis and atherosclerosis development in response to disturbed flow.

X-box binding protein 1 (XBP1) is a key signal transducer in endoplasmic reticulum stress response, and its potential role in the atherosclerosis development is unknown. This study aims to explore the impact of XBP1 on maintaining endothelial integrity related to atherosclerosis and to delineate the underlying mechanism. We found that XBP1 was highly expressed at branch points and areas of atherosclerotic lesions in the arteries of ApoE(-/-) mice, which was related to the severity of lesion development. In vitro study using human umbilical vein endothelial cells (HUVECs) indicated that disturbed flow increased the activation of XBP1 expression and splicing. Overexpression of spliced XBP1 induced apoptosis of HUVECs and endothelial loss from blood vessels during ex vivo cultures because of caspase activation and down-regulation of VE-cadherin resulting from transcriptional suppression and matrix metalloproteinase-mediated degradation. Reconstitution of VE-cadherin by Ad-VEcad significantly increased Ad-XBP1s-infected HUVEC survival. Importantly, Ad-XBP1s gene transfer to the vessel wall of ApoE(-/-) mice resulted in development of atherosclerotic lesions after aorta isografting. These results indicate that XBP1 plays an important role in maintaining endothelial integrity and atherosclerosis development, which provides a potential therapeutic target to intervene in atherosclerosis.

Embryonic stem cell differentiation into smooth muscle cells is mediated by Nox4-produced H2O2.

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.