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Dive into the research topics where Andriani Margariti is active.

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Featured researches published by Andriani Margariti.


American Journal of Physiology-cell Physiology | 2009

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

Qingzhong Xiao; Zhenling Luo; Anna Elena Pepe; Andriani Margariti; Lingfang Zeng; Qingbo Xu

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.


Journal of Biological Chemistry | 2013

XBP1 mRNA Splicing Triggers an Autophagic Response in Endothelial Cells through BECLIN-1 Transcriptional Activation

Andriani Margariti; Hongling Li; Ting Chen; Daniel Martin; Gema Vizcay-Barrena; Saydul Alam; Eirini Karamariti; Qingzhong Xiao; Anna Zampetaki; Zhongyi Zhang; Wen Wang; Zhixin Jiang; Chan Gao; Benyu Ma; Ye-Guang Chen; Gillian W. Cockerill; Yanhua Hu; Qingbo Xu; Lingfang Zeng

Background: Apoptosis and autophagy are two closely related systems that induce cell death. Results: X-box-binding protein 1 (XBP1) mRNA splicing regulates BECLIN-1 transcriptional activation, a fundamental player in the initiation of autophagy. Conclusion: XBP1 splicing induces an autophagic response in endothelial cells. Significance: XBP1 could be used as an important pharmacological target that can regulate the autophagic machinery and endothelial cell death. Sustained activation of X-box-binding protein 1 (XBP1) results in endothelial cell (EC) apoptosis and atherosclerosis development. The present study provides evidence that XBP1 mRNA splicing triggered an autophagic response in ECs by inducing autophagic vesicle formation and markers of autophagy BECLIN-1 and microtubule-associated protein 1 light chain 3β (LC3-βII). Endostatin activated autophagic gene expression through XBP1 mRNA splicing in an inositol-requiring enzyme 1α (IRE1α)-dependent manner. Knockdown of XBP1 or IRE1α by shRNA in ECs ablated endostatin-induced autophagosome formation. Importantly, data from arterial vessels from XBP1 EC conditional knock-out (XBP1eko) mice demonstrated that XBP1 deficiency in ECs reduced the basal level of LC3β expression and ablated response to endostatin. Chromatin immunoprecipitation assays further revealed that the spliced XBP1 isoform bound directly to the BECLIN-1 promoter at the region from nt −537 to −755. BECLIN-1 deficiency in ECs abolished the XBP1-induced autophagy response, whereas spliced XBP1 did not induce transcriptional activation of a truncated BECLIN-1 promoter. These results suggest that XBP1 mRNA splicing triggers an autophagic signal pathway through transcriptional regulation of BECLIN-1.


Arteriosclerosis, Thrombosis, and Vascular Biology | 2013

Adventitial Stem Cells in Vein Grafts Display Multilineage Potential That Contributes to Neointimal Formation

Yikuan Chen; Mei Mei Wong; Paola Campagnolo; Russell Simpson; Bernhard Winkler; Andriani Margariti; Yanhua Hu; Qingbo Xu

Objective—This study was designed to carry out the characterization of stem cells within the adventitia and to elucidate their functional role in the pathogenesis of vein graft atherosclerosis. Approach and Results—A mouse vein graft model was used to investigate the functional role of adventitial stem/progenitor cells on atherosclerosis. The adventitia of vein grafts underwent significant remodeling during early stages of vessel grafting and displayed markedly heterogeneous cell compositions. Immunofluorescence staining indicated a significant number of stem cell antigen-1–positive cells that were closely located to vasa vasorum. In vitro clonogenic assays demonstrated 1% to 11% of growing rates from adventitial cell cultures, most of which could be differentiated into smooth muscle cells (SMCs). These stem cell antigen-1–positive cells also displayed a potential to differentiate into adipogenic, osteogenic, or chondrogenic lineages in vitro. In light of the proatherogenic roles of SMCs in atherosclerosis, we focused on the functional roles of progenitor-SMC differentiation, in which we subsequently demonstrated that it was driven by direct interaction of the integrin/collagen IV axis. The ex vivo bioreactor system revealed the migratory capacity of stem cell antigen-1–positive progenitor cells into the vessel wall in response to stromal cell-derived factor-1. Stem cell antigen-1–positive cells that were applied to the outer layer of vein grafts showed enhanced atherosclerosis in apolipoprotein E–deficient mice, which contributed to ≈30% of neointimal SMCs. Conclusions—We demonstrate that during pathological conditions in vein grafting, the adventitia harbors stem/progenitor cells that can actively participate in the pathogenesis of vascular disease via differentiation into SMCs.


Arteriosclerosis, Thrombosis, and Vascular Biology | 2013

Sirolimus Stimulates Vascular Stem/Progenitor Cell Migration and Differentiation Into Smooth Muscle Cells via Epidermal Growth Factor Receptor/Extracellular Signal–Regulated Kinase/β-Catenin Signaling Pathway

Mei Mei Wong; Bernhard Winkler; Eirini Karamariti; Xiaocong Wang; Baoqi Yu; Russell Simpson; Ting Chen; Andriani Margariti; Qingbo Xu

Objective—Sirolimus-eluting stent therapy has achieved considerable success in overcoming coronary artery restenosis. However, there remain a large number of patients presenting with restenosis after the treatment, and the source of its persistence remains unclarified. Although recent evidence supports the contribution of vascular stem/progenitor cells in restenosis formation, their functional and molecular responses to sirolimus are largely unknown. Approach and Results—Using an established technique, vascular progenitor cells were isolated from adventitial tissues of mouse vessel grafts and purified with microbeads specific for stem cell antigen-1. We provide evidence that vascular progenitor cells treated with sirolimus resulted in an induction of their migration in both transwell and wound healing models, clearly mediated by CXCR4 activation. We confirmed the sirolimus-mediated increase of migration from the adventitial into the intima side using an ex vivo decellularized vessel scaffold, where they form neointima-like lesions that expressed high levels of smooth muscle cell (SMC) markers (SM-22&agr; and calponin). Subsequent in vitro studies confirmed that sirolimus can induce SMC but not endothelial cell differentiation of progenitor cells. Mechanistically, we showed that sirolimus-induced progenitor-SMC differentiation was mediated via epidermal growth factor receptor and extracellular signal–regulated kinase 1/2 activation that lead to &bgr;-catenin nuclear translocation. The ablation of epidermal growth factor receptor, extracellular signal–regulated kinase 1/2, or &bgr;-catenin attenuated sirolimus-induced SM-22&agr; promoter activation and SMC differentiation. Conclusions—These findings provide direct evidence of sirolimus-induced progenitor cell migration and differentiation into SMC via CXCR4 and epidermal growth factor receptor/extracellular signal–regulated kinase/&bgr;-catenin signal pathways, thus implicating a novel mechanism of restenosis formation after sirolimus-eluting stent treatment.


Arteriosclerosis, Thrombosis, and Vascular Biology | 2011

Chromobox Protein Homolog 3 Is Essential for Stem Cell Differentiation to Smooth Muscles In Vitro and in Embryonic Arteriogenesis

Qingzhong Xiao; Gang Wang; Xiaoke Yin; Zhenling Luo; Andriani Margariti; Lingfang Zeng; Manuel Mayr; Shu Ye; Qingbo Xu

Objective—Smooth muscle cell (SMC) differentiation is a critical process during cardiovascular formation and development, but the underlying molecular mechanism remains unclear. Methods and Results—Here we demonstrated that chromobox protein homolog 3 (Cbx3) is crucial for SMC differentiation from stem cells and that the chromodomain and chromoshadow domain of Cbx3 are responsible for Cbx3-induced SMC differentiation. Moreover, we identified that 4 amino acids (165 to 168) within the chromoshadow domain of Cbx3 are key elements for Cbx3 interaction with Dia-1- and Cbx3-induced SMC differentiation. Mechanistically, we found that Cbx3 mediates SMC differentiation through modulating serum response factor (SRF) recruitment to the promoters of SMC genes, in which the interaction between Cbx3 and Dia-1/SRF plays a crucial role in this process. Moreover, our in vivo study demonstrated that the misexpression of Cbx3 within neural crest cells of chick embryos resulted in the death of chick embryos at early stages because of the maldevelopment of branchial arch arteries. Conclusion—Our findings suggest that the interaction between Cbx3 and Dia-1/SRF is essential for SMC differentiation from stem cells and for the development of functional cardiovascular system.


Arteriosclerosis, Thrombosis, and Vascular Biology | 2014

Macrophages Control Vascular Stem/Progenitor Cell Plasticity Through Tumor Necrosis Factor-α–Mediated Nuclear Factor-κB Activation

Mei Mei Wong; Yikuan Chen; Andriani Margariti; Bernhard Winkler; Paola Campagnolo; Claire M.F. Potter; Yanhua Hu; Qingbo Xu

Objective— Vascular lineage differentiation of stem/progenitor cells can contribute to both tissue repair and exacerbation of vascular diseases such as in vein grafts. The role of macrophages in controlling vascular progenitor differentiation is largely unknown and may play an important role in graft development. This study aims to identify the role of macrophages in vascular stem/progenitor cell differentiation and thereafter elucidate the mechanisms that are involved in the macrophage- mediated process. Approach and Results— We provide in vitro evidence that macrophages can induce endothelial cell (EC) differentiation of the stem/progenitor cells while simultaneously inhibiting their smooth muscle cell differentiation. Mechanistically, both effects were mediated by macrophage-derived tumor necrosis factor-&agr; (TNF-&agr;) via TNF-&agr; receptor 1 and canonical nuclear factor-&kgr;B activation. Although the overexpression of p65 enhanced EC (or attenuated smooth muscle cell) differentiation, p65 or TNF-&agr; receptor 1 knockdown using lentiviral short hairpin RNA inhibited EC (or rescued smooth muscle cell) differentiation in response to TNF-&agr;. Furthermore, TNF-&agr;–mediated EC differentiation was driven by direct binding of nuclear factor-&kgr;B (p65) to specific VE-cadherin promoter sequences. Subsequent experiments using an ex vivo decellularized vessel scaffold confirmed an increase in the number of ECs and reduction in smooth muscle cell marker expression in the presence of TNF-&agr;. The lack of TNF-&agr; in a knockout mouse model of vein graft decreased endothelialization and significantly increased thrombosis formation. Conclusions— Our study highlights the role of macrophages in directing vascular stem/progenitor cell lineage commitment through TNF-&agr;–mediated TNF-&agr; receptor 1 and nuclear factor-&kgr;B activation that is likely required for endothelial repair in vascular diseases such as vein graft.


Heart | 2011

20 Direct reprogramming fibroblasts into endothelial cells

Andriani Margariti; Bernhard Winkler; Eirini Karamariti; Tsung-Neng Tsai; Lingfang Zeng; Yanhua Hu; Qingbo Xu

The generation of induced pluripotent stem (iPS) cells is a fascinated tool for regenerative medicine. However, the main restriction for iPS cell application is a risk of tumour development. In this study we established a new method to generate a partially induced stem (PiPS) cells by transferring four reprogramming factors (OCT4, SOX2, KLF4 and c-MYC) to human fibroblasts. PiPS cells did not form tumours in vivo, and have abilities to specifically differentiate into different cell lineages, such as neurons, adipocytes, osteocytes, chondrocytes as well as endothelial cells in response to define media and culture conditions. The PiPS-derived endothelial cells expressed a panel of endothelial markers at gene and protein levels and they formed vascular tubes in vitro and in vivo Matrigel plaque assays. To clarify the mechanism of PiPS cell differentiation into endothelial lineage, data from a series of experiments indicate a novel gene SETSIP was crucial in endothelial differentiation. SETSIP was expressed in parallel with endothelial genes, and its overexpression induced endothelial marker expression, while its downregulation by shRNA resulted in suppression of these genes. Interesting, this protein was translocated to the cell nucleus during endothelial differentiation, while ChIP assays showed that it bound direct to VE-cadherin promoter. Functionally, PiPS-derived endothelial cells displayed clearly endothelial properties in two animal models. When seeded on decellularised vessels in a special constructed bioreactor and implanted in SCID mice, the cells displayed well attachment, stabilisation, patency and typical vascular structure. Also, these cells showed endothelial engraftment to form typical vascular architecture when they were injected into the infarcted tissues in an ischaemia model. Thus, we developed a new method to generate PiPS cells from human fibroblasts that can differentiate into a variety of cell types without tumour risk, in which endothelial cells can be produced via OCT4-VEGF-SETSIP pathway useful for regenerating damaged tissue and vessels in vivo.


Heart | 2013

168 CIRCULATING ENDOTHELIAL PROGENITOR CELLS IN SMOKERS ARE DYSFUNCTIONAL DUE TO INCREASED DNA DAMAGE AND SENESCENCE

Koralia E. Paschalaki; Richard Starke; Yanhua Hu; Nicolas Mercado; Andriani Margariti; V G Gorgoulis; Peter J. Barnes; Anna M. Randi

Introduction Cardiovascular disease (CVD) is a major cause of death in smokers, particularly in patients with chronic obstructive pulmonary disease (COPD). Circulating endothelial progenitor cells (EPC) are required for endothelial homeostasis, and their dysfunction contributes to CVD. DNA damage has also been recognized as an important contributor to CVD. Our aim was to investigate whether EPC from smokers and COPD patients are dysfunctional, and to investigate the role of DNA damage pathways in mediating endothelial dysfunction in these patients. Methods To investigate EPC dysfunction in smokers, we isolated and expanded blood outgrowth endothelial cells (BOEC) from peripheral blood samples of healthy non-smokers, healthy smokers and COPD patients. The mononuclear fraction was placed in culture in the presence of endothelial growth factors and BOEC colonies appeared between days 7 and 24. BOEC colonies were expanded and used at passages 4 to 6 for all experiments. Endothelial senescence was measured by senescence-associated β-galactosidase (SA-β-Gal) activity. Expression of sirtuin (SIRT)-1 and of markers of senescence and DNA damage were measured by Western blotting and/or immunofluorescence confocal microscopy. SIRT1 activity was measured using a SIRT1 fluorescent activity assay kit. To investigate in vivo function, BOEC were labelled with Vybrant DiI Cell-Labelling Solution, mixed with Matrigel and injected subcutaneously into the back of NOD.CB17-Prkdcscid/NcrCrl mice. Seven days later, the mice were sacrificed and the plugs were harvested and cryosectioned. Results In vitro, BOEC from smokers and COPD patients showed increased DNA double-strand breaks (measured by γ-H2AX, 53BP1) and senescence (senescence associated-β-galactosidase activity, p16 and p21 levels) compared to non-smokers. Senescence negatively correlated with sirtuin-1 (SIRT1) expression and activity, a protein deacetylase that inhibits DNA damage and cellular senescence. Inhibition of DNA damage response by silencing of ataxia telangiectasia-mutated (ATM) kinase resulted in up-regulation of SIRT1 expression and decreased senescence. Interestingly, treatment of BOEC from COPD patients with the SIRT1 activator resveratrol or a selective ATM inhibitor rescued the senescent phenotype. Using the in vivo Matrigel plug angiogenesis assay, BOEC from COPD patients displayed reduced ability to form capillary-like structures and increased DNA damage, senescence and apoptosis (measured by 53BP1, p16, TUNEL and cleaved-caspase 3 staining) compared to non-smokers. Conclusions BOEC from smokers and COPD patients show reduced angiogenesis in vivo and display increased DNA damage and senescence, associated with reduced SIRT1 expression. These defects may contribute to endothelial dysfunction and cardiovascular events in smokers and COPD patients and could potentially constitute therapeutic targets for intervention.


Heart | 2013

185 SMOOTH MUSCLE CELLS DIFFERENTIATED FROM REPROGRAMMED FIBROBLASTS THROUGH DKK3 SIGNALLING ARE POTENT FOR TISSUE ENGINEERING OF VASCULAR GRAFTS

Qingbo Xu; Eirini Karamariti; Andriani Margariti; Yanhua Hu

Background Smooth muscle cells (SMCs) are a key component of tissue-engineered vessels. However, the sources by which they can be isolated are limited. The generation of induced pluripotent stem (iPS) cells is a useful tool for regenerative medicine. Nevertheless, the risk of tumor development of the aforementioned cells should be addressed before they can be used for clinical applications. During the reprogramming process a number of signal pathways are activated, which may lead to direct differentiation of specific cell lineages prior to the cells reaching the pluripotent state. Methods and Results We hypothesised that a large number of SMCs could be obtained by direct reprogramming of fibroblasts to SMCs. Therefore, we designed a combined protocol of reprogramming and differentiation in an attempt to achieve direct differentiation of fibroblasts to specific cell lineages. Human fibroblasts were shortly reprogrammed by overexpression of four reprogramming factors (OCT4, SOX2, KLF4 and c-MYC) and maintained in reprogramming media on a gelatin substrate for four days. These cells were defined as partially induced pluripotent stem (PiPS) cells. PiPS cells did not form tumors in vivo and differentiated into SMCs when seeded on a collagen IV substrate and maintained in differentiation media. The PiPS-SMCs expressed a panel of SMC markers such as SMA, SM22 and calponin at mRNA and protein levels. In order to elucidate the mechanism of PiPS cell differentiation into SMCs, data from a series of experiments indicated that the gene DKK3 was involved in this differentiation. DKK3 was expressed in parallel with SMC markers, while its overexpression or stimulation induced SMC marker expression. Furthermore, DKK3 silencing resulted in downregulation of SMC markers on both the mRNA and protein levels. Additional experiments revealed that the upregulation of SMC markers by DKK3 is mediated by interaction of DKK3 with the transmembrane receptor Kremen 1, potentiation of Wnt signalling and ultimately β-catenin translocation. Finally, PiPS-SMCs repopulated decellularised vessel grafts and ultimately gave rise to functional tissue-engineered vessels when combined with the previously established PiPS-endothelial cells, leading to increased survival of SCID mice after transplantation of the vessel as a vascular graft. Conclusion In the present study, we have developed a protocol to generate SMCs from PiPS cells through a DKK3 signalling pathway, which are useful for generating functional tissue-engineered vessels. These findings provide new insight into the mechanisms of SMC differentiation with vast therapeutic potential.


Heart | 2011

YIA 5 Splicing of HDAC7 modulates smooth muscle cell proliferation and neointima formation through nuclear β-catenin translocation

Boda Zhou; Andriani Margariti; Yanhua Hu; Qingbo Xu

Vascular smooth muscle cell (SMC) proliferation has an indispensable role in the pathogenesis of vascular disease, but the mechanism is not fully elucidated. The epigenetic enzyme histone deacetylase 7 (HDAC7) is involved in endothelial homeostasis and SMC differentiation but it is unknown whether it could have a role in SMC proliferation. In this study we sought to examine the effect of two HDAC7 isoforms on SMC proliferation and neointima formation. We demonstrated that overexpression of unspliced HDAC7 (HDAC7u) could suppress SMC proliferation through downregulation of cyclin D1 and cell cycle arrest, while spliced HDAC7 (HDAC7s) could not. SiRNA-mediated knockdown of HDAC7 increased SMC proliferation and induced nuclear translocation of β-catenin. Further experiments showed that only HDAC7u could bind to β-catenin and retain it in the cytoplasm. Reporter gene assay and reverse transcription PCR revealed a reduction of β-catenin activity in cells overexpressing HDAC7u, but not HDAC7s. Deletion studies indicated that the C-terminal region of HDAC7u is responsible for the interaction with β-catenin. However, the addition of amino acids to the N-terminus of HDAC7u disrupted the binding, further strengthening our hypothesis that HDAC7s does not interact with β-catenin. The growth factor PDGF-BB increased the splicing of HDAC7 while simultaneously decreasing the expression of HDAC7u. Importantly, in an animal model of femoral artery wire injury, we demonstrated that HDAC7 expression was elevated in a time-dependent manner at both mRNA and protein levels. Knockdown of HDAC7 by siRNA in the injured vessels aggravates neointima formation in comparison with control siRNA. Immunostaining for Ki67 of neointimal lesions displayed that proliferating cells were significantly increased in the vessel wall of HDAC7-siRNA treated group. Thus, our findings demonstrate that splicing of HDAC7 modulates SMC proliferation and neointima formation through β-catenin nuclear translocation, which provides a potential therapeutic target in vascular disease.

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Qingzhong Xiao

Queen Mary University of London

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