Expression of mitochondrial genes MT-ND1, MT-ND6, MT-CYB, MT-COI, MT-ATP6, and 12S/MT-RNR1 in colorectal adenopolyps.

Despite improvements in treatment strategies, colorectal cancer (CRC) still has high mortality rates. Most CRCs develop from adenopolyps via the adenoma-carcinoma sequence. A mechanism for inhibition of this sequence in individuals with a high risk of developing CRC is urgently needed. Differential studies of mitochondrial (mt) gene expressions in the progressive stages of CRC with villous architecture are warranted to reveal early risk assessments and new targets for chemoprevention of the disease. In the present study, reverse transcription-quantitative PCR (RT-qPCR) was used to determine the relative amount of the transcripts of six mt genes [MT-RNR1, MT-ND1, MT-COI, MT-ATP6, MT-ND6, and MT-CYB (region 648-15887)] which are involved in the normal metabolism of mitochondria. A total of 42 pairs of tissue samples obtained from colorectal adenopolyps, adenocarcinomas, and their corresponding adjacent normal tissues were examined. Additionally, electron transport chain (ETC), complexes I (NADH: ubiquinone oxidoreductase) and III (CoQH2-cytochrome C reductase), and carbonyl protein group contents were analyzed. Results indicate that there were differential expressions of the six mt genes and elevated carbonyl protein contents among the colorectal adenopolyps compared to their paired adjacent normal tissues (p < 0.05). The levels of complexes I and III were higher in tumor tissues relative to adjacent normal tissues. Noticeably, the expression of MT-COI was overexpressed in late colorectal carcinomas among all studied transcripts. Our data suggest that increased expressions in certain mt genes and elevated levels of ROS may potentially play a critical role in the colorectal tumors evolving from adenopolyps to malignant lesions.

Iroquois homeobox transcription factor (Irx5) promotes G1/S-phase transition in vascular smooth muscle cells by CDK2-dependent activation.

The Iroquois homeobox (Irx5) gene is essential in embryonic development and cardiac electrophysiology. Although recent studies have reported that IRX5 protein is involved in regulation of the cell cycle and apoptosis in prostate cancer cells, little is known about the role of IRX5 in the adult vasculature. Here we report novel observations on the role of IRX5 in adult vascular smooth muscle cells (VSMCs) during proliferation in vitro and in vivo. Comparative studies using primary human endothelial cells, VSMCs, and intact carotid arteries to determine relative expression of Irx5 in the peripheral vasculature demonstrate significantly higher expression in VSMCs. Sprague-Dawley rat carotid arteries were subjected to balloon catherization, and the presence of IRX5 was examined by immunohistochemistry after 2 wk. Results indicate markedly elevated IRX5 signal at 14 days compared with uninjured controls. Total RNA was isolated from injured and uninjured arteries, and Irx5 expression was measured by RT-PCR. Results demonstrate a significant increase in Irx5 expression at 3-14 days postinjury compared with controls. Irx5 genetic gain- and loss-of-function studies using thymidine and 5-bromo-2'-deoxyuridine incorporation assays resulted in modulation of DNA synthesis in primary rat aortic VSMCs. Quantitative RT-PCR results revealed modulation of cyclin-dependent kinase inhibitor 1B (p27(kip1)), E2F transcription factor 1 (E2f1), and proliferating cell nuclear antigen (Pcna) expression in Irx5-transduced VSMCs compared with controls. Subsequently, apoptosis was observed and confirmed by morphological observation, caspase-3 cleavage, and enzymatic activation compared with control conditions. Taken together, these results indicate that Irx5 plays an important role in VSMC G1/S-phase cell cycle checkpoint control and apoptosis.

Adipose-Derived Stem Cells Induce Angiogenesis via Microvesicle Transport of miRNA-31.

UNLABELLED: Cell secretion is an important mechanism for stem cell-based therapeutic angiogenesis, along with cell differentiation to vascular endothelial cells or smooth muscle cells. Cell-released microvesicles (MVs) have been recently implicated to play an essential role in intercellular communication. The purpose of this study was to explore the potential effects of stem cell-released MVs in proangiogenic therapy. We observed for the first time that MVs were released from adipose-derived stem cells (ASCs) and were able to increase the migration and tube formation of human umbilical vein endothelial cells (HUVECs). Endothelial differentiation medium (EDM) preconditioning of ASCs upregulated the release of MVs and enhanced the angiogenic effect of the released MVs in vitro. RNA analysis revealed that microRNA was enriched in ASC-released MVs and that the level of microRNA-31 (miR-31) in MVs was notably elevated upon EDM-preconditioning of MV-donor ASCs. Further studies exhibited that miR-31 in MVs contributed to the migration and tube formation of HUVECs, microvessel outgrowth of mouse aortic rings, and vascular formation of mouse Matrigel plugs. Moreover, factor-inhibiting HIF-1, an antiangiogenic gene, was identified as the target of miR-31 in HUVECs. Our findings provide the first evidence that MVs from ASCs, particularly from EDM-preconditioned ASCs, promote angiogenesis and the delivery of miR-31 may contribute the proangiogenic effect. SIGNIFICANCE: This study provides the evidence that microvesicles (MVs) from adipose-derived stem cells (ASCs), particularly from endothelial differentiation medium (EDM)-preconditioned ASCs, promote angiogenesis. An underlying mechanism of the proangiogenesis may be the delivery of microRNA-31 via MVs from ASCs to vascular endothelial cells in which factor-inhibiting HIF-1 is targeted and suppressed. The study findings reveal the role of MVs in mediating ASC-induced angiogenesis and suggest a potential MV-based angiogenic therapy for ischemic diseases.

Heme-Mediated Induction of CXCL10 and Depletion of CD34+ Progenitor Cells Is Toll-Like Receptor 4 Dependent.

Plasmodium falciparum infection can cause microvascular dysfunction, cerebral encephalopathy and death if untreated. We have previously shown that high concentrations of free heme, and C-X-C motif chemokine 10 (CXCL10) in sera of malaria patients induce apoptosis in microvascular endothelial and neuronal cells contributing to vascular dysfunction, blood-brain barrier (BBB) damage and mortality. Endothelial progenitor cells (EPC) are microvascular endothelial cell precursors partly responsible for repair and regeneration of damaged BBB endothelium. Studies have shown that EPC's are depleted in severe malaria patients, but the mechanisms mediating this phenomenon are unknown. Toll-like receptors recognize a wide variety of pathogen-associated molecular patterns generated by pathogens such as bacteria and parasites. We tested the hypothesis that EPC depletion during malaria pathogenesis is a function of heme-induced apoptosis mediated by CXCL10 induction and toll-like receptor (TLR) activation. Heme and CXCL10 concentrations in plasma obtained from malaria patients were elevated compared with non-malaria subjects. EPC numbers were significantly decreased in malaria patients (P < 0.02) and TLR4 expression was significantly elevated in vivo. These findings were confirmed in EPC precursors in vitro; where it was determined that heme-induced apoptosis and CXCL10 expression was TLR4-mediated. We conclude that increased serum heme mediates depletion of EPC during malaria pathogenesis.

MicroRNA-27 (miR-27) targets prohibitin and impairs adipocyte differentiation and mitochondrial function in human adipose-derived stem cells.

Prohibitin (PHB) has been reported to play a crucial role in adipocyte differentiation and mitochondrial function. However, the regulative mechanism of PHB during adipogenesis remains unclear. In this study, we determined that the levels of both microRNA (miR)-27a and miR-27b were down-regulated following adipogenic induction of human adipose-derived stem cells, whereas the mRNA level of PHB was up-regulated. Overexpression of miR-27a or miR-27b inhibited PHB expression and adipocyte differentiation. Using PHB 3'-UTR luciferase reporter assay, we observed that miR-27a and miR-27b directly targeted PHB in human adipose-derived stem cells. A compensation of PHB partially restored the adipogenesis inhibited by miR-27. Moreover, we demonstrated the novel finding that ectopic expression of miR-27a or miR-27b impaired mitochondrial biogenesis, structure integrity, and complex I activity accompanied by excessive reactive oxygen species production. Our data suggest that miR-27 is an anti-adipogenic microRNA partly by targeting PHB and impairing mitochondrial function. Pharmacological modulation of miR-27 function may provide a new therapeutic strategy for the treatment of obesity.

Neurotrophic effects of leukemia inhibitory factor on neural cells derived from human embryonic stem cells.

Various growth factor cocktails have been used to proliferate and then differentiate human neural progenitor (NP) cells derived from embryonic stem cells (ESC) for in vitro and in vivo studies. However, the cytokine leukemia inhibitory factor (LIF) has been largely overlooked. Here, we demonstrate that LIF significantly enhanced in vitro survival and promoted differentiation of human ESC-derived NP cells. In NP cells, as well as NP-derived neurons, LIF reduced caspase-mediated apoptosis and reduced both spontaneous and H2O2-induced reactive oxygen species in culture. In vitro, NP cell proliferation and the yield of differentiated neurons were significantly higher in the presence of LIF. In NP cells, LIF enhanced cMyc phosphorylation, commonly associated with self-renewal/proliferation. Also, in differentiating NP cells LIF activated the phosphoinositide 3-kinase and signal transducer and activator of transcription 3 pathways, associated with cell survival and reduced apoptosis. When differentiated in LIF+ media, neurite outgrowth and ERK1/2 phosphorylation were potentiated together with increased expression of gp130, a component of the LIF receptor complex. NP cells, pretreated in vitro with LIF, were effective in reducing infarct volume in a model of focal ischemic stroke but LIF did not lead to significantly improved initial NP cell survival over nontreated NP cells. Our results show that LIF signaling significantly promotes human NP cell proliferation, survival, and differentiation in vitro. Activated LIF signaling should be considered in cell culture expansion systems for future human NP cell-based therapeutic transplant studies.

SUV39H1 orchestrates temporal dynamics of centromeric methylation essential for faithful chromosome segregation in mitosis.

Histone methylation performs multiple functions such as DNA replication, transcription regulation, heterochromatin formation, and chromatin condensation. How this methylation gradient is orchestrated in the centromere during chromosome segregation is not known. Here we examine the temporal dynamics of protein methylation in the centromere by SUV39H1 methyltransferase, a key mitotic regulator, using fluorescence resonance energy transfer-based sensors in living HeLa cells and immunofluorescence of native SUV39H1 substrates. A quantitative analysis of methylation dynamics, using centromere-targeted sensors, reveals a temporal change during chromosome segregation. These dynamics result in an accurate chromosome congression to and alignment at the equator as an inhibition of methylation dynamics using SUV39H1 inhibitor perturbs chromosome congression in living HeLa cells. Surprisingly, this inhibition of methylation results in a brief increase in Aurora B kinase activity and an enrichment of microtubule depolymerase MCAK in the centromere with a concomitant kinetochore-microtubule destabilization and a reduced tension across the sister kinetochores with ultimate chromosome misalignments. We reason that SUV39H1 generates a gradient of methylation marks at the kinetochore that provides spatiotemporal information essential for accurate chromosome segregation in mitosis.

Endoglin is a novel endothelial cell specification gene.

Endothelial cells (EC) are important in vasculogenesis and organogenesis during development and in the pathogenesis of cancer and cardiovascular diseases. However, few EC specification factors are known and primary EC production remains inefficient. Based on recent studies implicating endoglin (Eng) in early vascular development and angiogenesis, we hypothesized that Eng may be an EC specification gene. Mouse embryonic stem cells (ESC) were treated with recombinant Eng or a plasmid expressing the Eng ORF, and differentiated in the presence or absence of bone morphogenic protein 4 (BMP4). Expression of the mesoderm and EC marker genes, the known mediators of EC specification and their downstream targets was monitored by quantitative PCR, western blot, immunocytochemistry, and flow cytometry. Functionality of the differentiated EC was assessed by in vitro angiogenesis assay and the induction of Icam1 expression in response to TNF-α treatment. Both recombinant Eng and forced Eng expression increased the number of functional EC expressing the EC marker genes VE-cadherin, vWF, and Tie2, and enhanced the effect of BMP4. The Eng-induced EC differentiation was independent of known mediators of EC specification such as Indian Hedgehog (IHH) and BMP4 or of BMP4/Smad1/5/8 signaling. These studies suggest that Eng is a novel EC specification gene.

Inhibition of DNA methyltransferases and histone deacetylases induces bone marrow-derived multipotent adult progenitor cells to differentiate into endothelial cells.

INTRODUCTION: Endothelial dysfunction plays a critical role in the pathogenesis of cardiovascular diseases and cancer. Bone marrow-derived multipotent adult progenitor cells (MAPC) have the potential to differentiate, at the single cell level, toward the three embryonic germ layers and may be the progenitors of the other tissue-specific stem cells. However, molecular mechanisms of endothelial differentiation from MAPC have not been defined. The importance of epigenetic changes such as DNA methylation and histone acetylation in gene regulatory networks during embryonic stem cell (ESC) differentiation has been documented. We postulated that endothelial cell (EC) differentiation from MAPC could be enhanced by inhibiting DNA methylation and histone deacetylation, reversing the repression of genes that specify EC fate. METHODS: MAPCs were derived from rat bone marrow and differentiated into EC by vascular endothelial growth factor (VEGF) treatment in the presence or absence of the specific DNA methyltransferase (DNMT) inhibitor 5'-aza-2'-deoxycytidine (aza-dC) and the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). Expression of the endothelial marker genes was assessed by real time quantitative PCR and angiogenic potential of the differentiated EC was assessed by analysis of vascular network formation on fibronectin. RESULTS: Both aza-dC and TSA induced at least a three-fold increase in the expression of the EC marker genes VE-cadherin, vWF, and Flk1. This increase was also observed in the presence of the EC differentiation inducer VEGF, suggesting that factors other than VEGF mediate the response to the epigenetic agents. Both DNMT and HDAC inhibition stimulated vascular network formation. CONCLUSION: Epigenetic therapy holds a potential in inducing self-repair, vascular tissue regeneration, controlling angiogenesis and endothelial dysfunction.

DNA methyltransferase inhibition induces mouse embryonic stem cell differentiation into endothelial cells.

Understanding endothelial cell (EC) differentiation is a step forward in tissue engineering, controlling angiogenesis, and endothelial dysfunction. We hypothesized that epigenetic activation of EC lineage specification genes is an important mediator of embryonic stem cell (ESC) differentiation into EC. Mouse ESC was differentiated by removing leukemia inhibitory factor (LIF) from the maintenance media in the presence or absence of the specific DNA methyltransferase (DNMT) inhibitor 5'-aza-2'-deoxycytidine (aza-dC). Expression of EC specification and marker genes was monitored by quantitative PCR, western, immunocytochemistry, and flow cytometry. Functionality of differentiated EC was assessed by angiogenesis assay. The methylation status in the proximal promoter CpGs of the mediators of EC differentiation VEGF-A, BMP4, and EPAS-1 as well as of the mature EC marker VE-cadherin was determined by bisulfite sequencing. ESC differentiation resulted in repression of OCT4 expression in both the absence and presence of aza-dC treatment. However, significant increase in angiogenesis and expression of the mediators of EC differentiation and EC-specific genes was only observed in aza-dC-treated cells. The DNMT inhibition-mediated increase in EC specification and marker gene expression was not associated with demethylation of these genes. These studies suggest that DNMT inhibition is an efficient inducer of EC differentiation from ESC.

Activation of p300 histone acetyltransferase activity is an early endothelial response to laminar shear stress and is essential for stimulation of endothelial nitric-oxide synthase mRNA transcription.

Previous studies have shown that the acute stimulation of endothelial nitric-oxide synthase (eNOS) mRNA transcription by laminar shear stress is dependent on nuclear factor kappa B (NFkappaB) subunits p50 and p65 binding to a shear stress response element (SSRE) in the human eNOS promoter and that mutation of the SSRE abrogates the shear-stimulated increase in eNOS promoter activity. In the present study, we found that although shear markedly increased eNOS mRNA, the increase in nuclear translocation of p50 and p65 caused by shear was only 2-fold, suggesting that shear has additional effects on NFkappaB cofactor activity beyond nuclear translocation. Chromatin immunoprecipitation assays showed that virtually no p50 or p65 was bound to the eNOS promoter at base line but that shear increased the binding of these subunits to the human eNOS SSRE by 10- to 20-fold. Co-immunoprecipitation studies demonstrated during the first 30 min of shear p300 bound to p65. Shear also increased p300 histone acetyltransferase (HAT) activity by 2.5-fold and increased acetylation of p65. The increase in eNOS mRNA caused by shear was completely blocked by pharmacological inhibition of p300/HAT activity with curcumin or by p300 small interfering RNA. Chromatin immunoprecipitation assays also showed that shear stimulated acetylation of histones 3 and 4 at the region of the eNOS promoter SSRE and extended 3' toward the eNOS coding region. This was associated with opening of chromatin at the SSRE. In conclusion, these studies reveal a previously unknown role of p300/HAT activation as a very early response to shear that is essential for increasing eNOS mRNA levels.

Cardiac peroxisome proliferator-activated receptor gamma is essential in protecting cardiomyocytes from oxidative damage.

OBJECTIVES: Peroxisome proliferator-activated receptors (PPAR) alpha and beta/delta are essential transcriptional regulators of fatty acid oxidation in the heart. However, little is known about the roles of PPARgamma in the heart. The present study is to investigate in vivo role(s) of PPARgamma in the heart. METHODS: A Cre-loxP mediated cardiomyocyte-restricted PPARgamma knockout line was investigated. In these mice, exon 1 and 2 of PPARgamma were targeted to eliminate PPARgamma from cardiomyocytes. RESULTS: PPARgamma null mice exhibited pathological changes around 3 months of age, featuring progressive cardiac hypertrophy with mitochondrial oxidative damage. Most mice died from dilated cardiomyopathy. Cardiac expression of Sod2 (encoding manganese superoxide dismutase; MnSOD), a mitochondrial antioxidant enzyme was downregulated both in transcript and protein levels in cardiac samples in PPARgamma knockout mice independent of pathological changes. Promoter analyses revealed that Sod2 is a target gene of PPARgamma. Consequently, myocardial superoxide content in PPARgamma knockout mice was increased, leading to extensive oxidative damage. Treatment with a SOD mimetic compound, MnTBAP, prevented superoxide-induced cardiac pathological changes in PPARgamma knockout mice. CONCLUSIONS: The present study demonstrates that PPARgamma is critical to myocardial redox homeostasis. These findings should provide new insights into understanding the roles of PPARgamma in the heart.

Interleukin-1 beta-induced Id2 gene expression is mediated by Egr-1 in vascular smooth muscle cells.

OBJECTIVE: Id2 (inhibitor of DNA-binding 2), a member of the helix-loop-helix family of transcription regulators, plays important roles in cell proliferation and differentiation. Recent reports have documented that Id2 is up-regulated during vascular lesion formation and overexpression of Id2 promotes vascular smooth muscle cell (VSMC) proliferation. However, the transcriptional regulation of Id2 gene expression in VSMC remains unexplored. METHODS AND RESULTS: Using Northern- and Western-blot analyses, we documented that interleukin-1beta (IL-1beta) induced Id2 gene expression in VSMC in a time- and dose-dependent manner. Overexpression of early growth response-1 (Egr-1) in VSMC induced Id2 expression while IL-1beta-induced Id2 expression was abrogated in VSMC by the Egr-1 repressor, NGFI-A binding protein 2 (NAB2), expressed from an adenovirus. Overexpression of Egr-1 transactivated the Id2 promoter in reporter assays dependent on the presence of intact putative Egr-1 binding sites as determined by mutagenesis. Finally, electrophoretic mobility shift assays (EMSA) demonstrated that the Egr-1 protein can bind the Egr-1 sites derived from the human Id2 promoter in vitro and chromatin immunoprecipitation identified the putative Egr-1 site between -723 to -712 as the functional Egr-1 binding site in vivo. CONCLUSIONS: Our data demonstrate that IL-1beta-induced Id2 expression in VSMC is mediated by the transcription factor Egr-1 in VSMC.