Sifeng Chen

Ecto-5′-nucleotidase promotes invasion, migration and adhesion of human breast cancer cells

PurposeAssociated with many molecules, metastasis includes cell adhesion to extracellular matrix, migration towards specific direction and invasion into local vessel of distant organs. The purpose of the present study was to evaluate the role of ecto-5′-nucleotidase (eN, ecto-5-NT, CD73) generated extracellular adenosine in biologically malignant behaviors of human breast cancer cell lines.Materials and methodsTwo human breast cancer cell lines, T-47D with lower expression of CD73 and MB-MDA-231 with higher expression of CD73, were used to investigate the functions of CD73. The effects of CD73 over-expression on invasion, migration and adhesion were observed in T-47D transfected with pcDNA-NT5E plasmid. The effects of specific CD73 inhibitor, α, ß-methylene ADP (APCP), were observed in MB-MDA-231 cells.ResultsThe results showed CD-73 overexpression increased invasion, migration and adhesion to ECM of the pcDNA-NT5E transfected T-47D cells compared to the saline and mock vector controls. The increased cell mobility of CD-73-overexpressed T-47D cells was blocked by APCP. Adenosine increased the mobility of wild type T-47D cells. APCP inhibited the mobility of the MB-MDA-231 cells.ConclusionTaken together, our results indicated that CD73 may facilitate the adhesion, migration and invasion of human breast cancer cells through its enzyme activity of generating adenosine. This study provided a possibly molecular mechanism of metastasis of breast carcinoma.

RNA interference of ecto-5′-nucleotidase (CD73) inhibits human breast cancer cell growth and invasion

Metastasis is a leading cause of mortality and morbidity in breast cancer. Recently, dramatic overexpression of ecto-5′-nucleotidase (CD73), a glycosylphosphatidylinositol-anchored cell surface protein has been found in estrogen receptor-negative [ER (−)] breast cancer cell lines and in clinical samples. In this study, CD73 small interfering RNA (siRNA) plasmid was constructed and stably transfected into breast cancer cell MB-MDA-231 to determine the role of CD73 in breast cancer metastasis and the possible mechanism. Our study demonstrates that CD73 siRNA effectively inhibits CD73 gene expression at mRNA and protein level in MB-MDA-231 cells, leading to in vivo and in vitro growth suppression, prevention of adhesion to extracellular matrix (ECM), and inhibition of invasion and migration. These properties correlate with inhibition of matrix metalloproteinase (MMP)-2 and MMP-9 expression and activity as well as reduction of epidermal growth factor receptor (EGFR) expression. Demonstration of the role of CD73 in breast cancer may lead to new targeted therapies for breast cancer.

Overexpression of mitochondrial cholesterol delivery protein, StAR, decreases intracellular lipids and inflammatory factors secretion in macrophages

Hyperlipidemia is one of the most important risk factors for atherosclerosis. This can be amplified by a localized inflammatory response mediated by macrophages. Macrophages are capable of taking up excess cholesterol, and it is well known that delivery of cholesterol to the mitochondria by steroidogenic acute regulatory (StAR) protein is the rate-limiting step for cholesterol degradation in the liver. It has also been shown that overexpression of StAR in hepatocytes dramatically increases the amount of regulatory oxysterols in the nucleus, which play an important role in the maintenance of intracellular lipid homeostasis. The goal of the present study was to determine whether StAR plays a similar role in macrophages. We have found that overexpression of StAR in human THP-1 monocyte-derived macrophages decreases intracellular lipid levels, activates liver X receptor alpha (LXRalpha) and proliferation peroxysome activator receptor gamma (PPARgamma), and increases ABCG1 and CYP27A1 expression. Furthermore, it reduces the secretion of inflammatory factors, and prevents apoptosis. These results suggest that StAR delivers cholesterol to mitochondria where regulatory oxysterols are generated. Regulatory oxysterols can in turn activate nuclear receptors, which increase expression of cholesterol efflux transporters, and decrease secretion of inflammatory factors. These effects can prevent macrophage apoptosis. These results imply a potential role of StAR in the prevention of atherosclerosis.

Bach1 Represses Wnt/β-Catenin Signaling and Angiogenesis

RATIONALE Wnt/β-catenin signaling has an important role in the angiogenic activity of endothelial cells (ECs). Bach1 is a transcription factor and is expressed in ECs, but whether Bach1 regulates angiogenesis is unknown. OBJECTIVE This study evaluated the role of Bach1 in angiogenesis and Wnt/β-catenin signaling. METHODS AND RESULTS Hind-limb ischemia was surgically induced in Bach1(-/-) mice and their wild-type littermates and in C57BL/6J mice treated with adenoviruses coding for Bach1 or GFP. Lack of Bach1 expression was associated with significant increases in perfusion and vascular density and in the expression of proangiogenic cytokines in the ischemic hindlimb of mice, with enhancement of the angiogenic activity of ECs (eg, tube formation, migration, and proliferation). Bach1 overexpression impaired angiogenesis in mice with hind-limb ischemia and inhibited Wnt3a-stimulated angiogenic response and the expression of Wnt/β-catenin target genes, such as interleukin-8 and vascular endothelial growth factor, in human umbilical vein ECs. Interleukin-8 and vascular endothelial growth factor were responsible for the antiangiogenic response of Bach1. Immunoprecipitation and GST pull-down assessments indicated that Bach1 binds directly to TCF4 and reduces the interaction of β-catenin with TCF4. Bach1 overexpression reduces the interaction between p300/CBP and β-catenin, as well as β-catenin acetylation, and chromatin immunoprecipitation experiments confirmed that Bach1 occupies the TCF4-binding site of the interleukin-8 promoter and recruits histone deacetylase 1 to the interleukin-8 promoter in human umbilical vein ECs. CONCLUSIONS Bach1 suppresses angiogenesis after ischemic injury and impairs Wnt/β-catenin signaling by disrupting the interaction between β-catenin and TCF4 and by recruiting histone deacetylase 1 to the promoter of TCF4-targeted genes.

Inhibition of Myocardial Ischemia/Reperfusion Injury by Exosomes Secreted from Mesenchymal Stem Cells.

Exosomes secreted by mesenchymal stem cells have shown great therapeutic potential in regenerative medicine. In this study, we performed meta-analysis to assess the clinical effectiveness of using exosomes in ischemia/reperfusion injury based on the reports published between January 2000 and September 2015 and indexed in the PUBMED and Web of Science databases. The effect of exosomes on heart function was evaluated according to the following parameters: the area at risk as a percentage of the left ventricle, infarct size as a percentage of the area at risk, infarct size as a percentage of the left ventricle, left ventricular ejection fraction, left ventricular fraction shortening, end-diastolic volume, and end-systolic volume. Our analysis indicated that the currently available evidence confirmed the therapeutic potential of mesenchymal stem cell-secreted exosomes in the improvement of heart function. However, further mechanistic studies, therapeutic safety, and clinical trials are required for optimization and validation of this approach to cardiac regeneration after ischemia/reperfusion injury.

CCND2 Overexpression Enhances the Regenerative Potency of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Remuscularization of Injured Ventricle

Rationale: The effectiveness of transplanted, human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) for treatment of ischemic myocardial injury is limited by the exceptionally low engraftment rate. Objective: To determine whether overexpression of the cell cycle activator CCND2 (cyclin D2) in hiPSC-CMs can increase the graft size and improve myocardial recovery in a mouse model of myocardial infarction by increasing the proliferation of grafted cells. Methods and Results: Human CCND2 was delivered to hiPSCs via lentiviral-mediated gene transfection. In cultured cells, markers for cell cycle activation and proliferation were ≈3- to 7-folds higher in CCND2-overexpressing hiPSC-CMs (hiPSC-CCND2OECMs) than in hiPSC-CMs with normal levels of CCND2 (hiPSC-CCND2WTCMs; P<0.01). The pluripotent genes (Oct 4, Sox2, and Nanog) decrease to minimal levels and undetectable levels at day 1 and 10 after differentiating to CMs. In the mouse myocardial infarction model, cardiac function, infarct size, and the number of engrafted cells were similar at week 1 after treatment with hiPSC-CCND2OECMs or hiPSC-CCND2WTCMs but was about tripled in hiPSC-CCND2OECM–treated than in hiPSC-CCND2WTCM–treated animals at week 4 (P<0.01). The cardiac function and infarct size were significantly better in both cell treatment groups’ hearts than in control hearts, which was most prominent in hiPSC-CCND2OECM–treated animals (P<0.05, each). No tumor formation was observed in any hearts. Conclusions: CCND2 overexpression activates cell cycle progression in hiPSC-CMs that results in a significant enhanced potency for myocardial repair as evidenced by remuscularization of injured myocardium. This left ventricular muscle regeneration and increased angiogenesis in border zone are accompanied by a significant improvement of left ventricular chamber function.

Clock upregulates intercellular adhesion molecule-1 expression and promotes mononuclear cells adhesion to endothelial cells.

Clock is a basic helix-loop-helix (bHLH) transcription factor that plays important role in circadian rhythms of various physiological functions. Previous study showed that the expression of intercellular adhesion molecule-1 (ICAM-1) was reduced in the liver tissues of Clock mutant mice. However, how Clock regulates ICAM-1 expression and whether Clock affects cell adhesion function remain unknown. In the present study, we found that exogenous expression of Clock upregulated the gene expressions of ICAM-1 and other adhesion-related genes including VCAM1 and CCL-2, and increased the transcriptional activity of ICAM-1 in mouse brain microvascular endothelial cell lines. In contrast, loss of Clock decreased these gene expressions and ICAM-1 transcriptional activity. Chromatin immunoprecipitation (ChIP) assay revealed that Clock binds to the E-box-like enhancer of ICAM-1 gene. ICAM-1 gene showed rhythmic expression in endothelial cells after serum shock in vitro, suggesting ICAM-1 may be a Clock-controlled gene. Clock regulates the adhesion of mononuclear cells to endothelial cells via ICAM-1. Together, our findings show that Clock is a positive regulator of ICAM-1, and promotes the adhesion of mononuclear cells to endothelial cells.

Nox2 contributes to the arterial endothelial specification of mouse induced pluripotent stem cells by upregulating Notch signaling

Reactive oxygen species (ROS) have a crucial role in stem-cell differentiation; however, the mechanisms by which ROS regulate the differentiation of stem cells into endothelial cells (ECs) are unknown. Here, we determine the role of ROS produced by NADPH oxidase 2 (Nox2) in the endothelial-lineage specification of mouse induced-pluripotent stem cells (miPSCs). When wild-type (WT) and Nox2-knockout (Nox2−/−) miPSCs were differentiated into ECs (miPSC-ECs), the expression of endothelial markers, arterial endothelial markers, pro-angiogenic cytokines, and Notch pathway components was suppressed in the Nox2−/− cells but increased in both WT and Nox2−/− miPSCs when Nox2 expression was upregulated. Higher levels of Nox2 expression increased Notch signaling and arterial EC differentiation, and this increase was abolished by the inhibition of ROS generation or by the silencing of Notch1 expression. Nox2 deficiency was associated with declines in the survival and angiogenic potency of miPSC-ECs, and capillary and arterial density were lower in the ischemic limbs of mice after treatment with Nox2−/− miPSC-ECs than WT miPSC-EC treatment. Taken together, these observations indicate that Nox2-mediated ROS production promotes arterial EC specification in differentiating miPSCs by activating the Notch signaling pathway and contributes to the angiogenic potency of transplanted miPSC-derived ECs.

Human induced pluripotent stem cells derived endothelial cells mimicking vascular inflammatory response under flow

Endothelial cells (ECs) have great potential in vascular diseases research and regenerative medicine. Autologous human ECs are difficult to acquire in sufficient numbers in vitro, and human induced pluripotent stem cells (iPSCs) offer unique opportunity to generate ECs for these purposes. In this work, we present a new and efficient method to simply differentiate human iPSCs into functional ECs, which can respond to physiological level of flow and inflammatory stimulation on a fabricated microdevice. The endothelial-like cells were differentiated from human iPSCs within only one week, according to the inducing development principle. The expression of endothelial progenitor and endothelial marker genes (GATA2, RUNX1, CD34, and CD31) increased on the second and fourth days after the initial inducing process. The differentiated ECs exhibited strong expression of cells-specific markers (CD31 and von Willebrand factor antibody), similar to that present in human umbilical vein endothelial cells. In addition, the hiPSC derived ECs were able to form tubular structure and respond to vascular-like flow generated on a microdevice. Furthermore, the human induced pluripotent stem cell-endothelial cells (hiPSC-ECs) pretreated with tumor necrosis factor (TNF-α) were susceptible to adhesion to human monocyte line U937 under flow condition, indicating the feasibility of this hiPSCs derived microsystem for mimicking the inflammatory response of endothelial cells under physiological and pathological process.

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

Reactive oxygen species (ROS) and redox homeostasis have a pivotal role in the maintenance of stem cell pluripotency and in stem cell self‐renewal; however, the mechanisms by which ROS regulate the self‐renewal of stem cells have not been thoroughly studied. Here, we evaluated the role of the ROS produced by NADPH oxidase 2 (Nox2) and NADPH oxidase 4 (Nox4) in the self‐renewal and stemness of murine induced‐pluripotent stem cells (miPSCs). Targeted silencing of Nox2 or Nox4 reduced both NADPH oxidase activity and intracellular ROS levels, as well as alkaline phosphatase activity, the total number of miPSCs, the expression of insulin‐like growth factor‐1 (IGF‐1), IGF‐1 receptor, and the phosphorylation of extracellular signal regulated kinase (ERK) 1/2. Nox2/Nox4 overexpression or low, nontoxic concentration of H2O2 increased cell proliferation in miPSCs. Furthermore, expression of the stemness genes Sox2 and Oct4 was lower in Nox2/Nox4‐deficient miPSCs, and higher in Nox2/Nox4‐overexpressing miPSCs, than in miPSCs with normal levels of Nox2/Nox4 expression. Collectively, these results suggest that Nox2‐ and Nox4‐derived ROS contribute to stem cell pluripotency maintenance and self‐renewal.