Antonis K. Hatzopoulos

A family of octamer-specific proteins present during mouse embryogenesis: Evidence for germline-specific expression of an Oct factor.

We have analysed various adult organs and different developmental stages of mouse embryos for the presence of octamer‐binding proteins. A variety of new octamer‐binding proteins were identified in addition to the previously described Oct1 and Oct2. Oct1 is ubiquitously present in murine tissues, in agreement with cell culture data. Although Oct2 has been described as a B‐cell‐specific protein, similar complexes were also found with extracts from brain, kidney, embryo and sperm. In embryo and brain at least two other proteins, Oct3 and Oct7, are present. A new microextraction procedure allowed the detection of two maternally expressed octamer‐binding proteins, Oct4 and Oct5. Both proteins are present in unfertilized oocytes and embryonic stem cells, the latter containing an additional protein, Oct6. Whereas Oct4 was not found in sperm or testis, it is expressed in male and female primordial germ cells. Therefore Oct4 expression is specific for the female germline at later stages of germ cell development. Our results indicate that a family of octamer‐binding proteins is present during mouse development and is differentially expressed during early embryogenesis. Protease clipping experiments of Oct4 and Oct1 suggest that both proteins contain similar DNA‐binding domains.

Essential Role for Mitochondrial Thioredoxin Reductase in Hematopoiesis, Heart Development, and Heart Function

ABSTRACT Oxygen radicals regulate many physiological processes, such as signaling, proliferation, and apoptosis, and thus play a pivotal role in pathophysiology and disease development. There are at least two thioredoxin reductase/thioredoxin/peroxiredoxin systems participating in the cellular defense against oxygen radicals. At present, relatively little is known about the contribution of individual enzymes to the redox metabolism in different cell types. To begin to address this question, we generated and characterized mice lacking functional mitochondrial thioredoxin reductase (TrxR2). Ubiquitous Cre-mediated inactivation of TrxR2 is associated with embryonic death at embryonic day 13. TrxR2 TrxR2−/− minus;/TrxR2−/− minus; embryos are smaller and severely anemic and show increased apoptosis in the liver. The size of hematopoietic colonies cultured ex vivo is dramatically reduced. TrxR2-deficient embryonic fibroblasts are highly sensitive to endogenous oxygen radicals when glutathione synthesis is inhibited. Besides the defect in hematopoiesis, the ventricular heart wall of TrxR2 TrxR2−/− minus;/TrxR2−/− minus; embryos is thinned and proliferation of cardiomyocytes is decreased. Cardiac tissue-restricted ablation of TrxR2 results in fatal dilated cardiomyopathy, a condition reminiscent of that in Keshan disease and Friedreichs ataxia. We conclude that TrxR2 plays a pivotal role in both hematopoiesis and heart function.

Effect of transendocardial delivery of autologous bone marrow mononuclear cells on functional capacity, left ventricular function, and perfusion in chronic heart failure: the FOCUS-CCTRN trial.

CONTEXT Previous studies using autologous bone marrow mononuclear cells (BMCs) in patients with ischemic cardiomyopathy have demonstrated safety and suggested efficacy. OBJECTIVE To determine if administration of BMCs through transendocardial injections improves myocardial perfusion, reduces left ventricular end-systolic volume (LVESV), or enhances maximal oxygen consumption in patients with coronary artery disease or LV dysfunction, and limiting heart failure or angina. DESIGN, SETTING, AND PATIENTS A phase 2 randomized double-blind, placebo-controlled trial of symptomatic patients (New York Heart Association classification II-III or Canadian Cardiovascular Society classification II-IV) with a left ventricular ejection fraction of 45% or less, a perfusion defect by single-photon emission tomography (SPECT), and coronary artery disease not amenable to revascularization who were receiving maximal medical therapy at 5 National Heart, Lung, and Blood Institute-sponsored Cardiovascular Cell Therapy Research Network (CCTRN) sites between April 29, 2009, and April 18, 2011. INTERVENTION Bone marrow aspiration (isolation of BMCs using a standardized automated system performed locally) and transendocardial injection of 100 million BMCs or placebo (ratio of 2 for BMC group to 1 for placebo group). MAIN OUTCOME MEASURES Co-primary end points assessed at 6 months: changes in LVESV assessed by echocardiography, maximal oxygen consumption, and reversibility on SPECT. Phenotypic and functional analyses of the cell product were performed by the CCTRN biorepository core laboratory. RESULTS Of 153 patients who provided consent, a total of 92 (82 men; average age: 63 years) were randomized (n = 61 in BMC group and n = 31 in placebo group). Changes in LVESV index (-0.9 mL/m(2) [95% CI, -6.1 to 4.3]; P = .73), maximal oxygen consumption (1.0 [95% CI, -0.42 to 2.34]; P = .17), and reversible defect (-1.2 [95% CI, -12.50 to 10.12]; P = .84) were not statistically significant. There were no differences found in any of the secondary outcomes, including percent myocardial defect, total defect size, fixed defect size, regional wall motion, and clinical improvement. CONCLUSION Among patients with chronic ischemic heart failure, transendocardial injection of autologous BMCs compared with placebo did not improve LVESV, maximal oxygen consumption, or reversibility on SPECT. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00824005.

Effect of Intracoronary Delivery of Autologous Bone Marrow Mononuclear Cells 2 to 3 Weeks Following Acute Myocardial Infarction on Left Ventricular Function The LateTIME Randomized Trial

CONTEXT Clinical trial results suggest that intracoronary delivery of autologous bone marrow mononuclear cells (BMCs) may improve left ventricular (LV) function when administered within the first week following myocardial infarction (MI). However, because a substantial number of patients may not present for early cell delivery, the efficacy of autologous BMC delivery 2 to 3 weeks post-MI warrants investigation. OBJECTIVE To determine if intracoronary delivery of autologous BMCs improves global and regional LV function when delivered 2 to 3 weeks following first MI. DESIGN, SETTING, AND PATIENTS A randomized, double-blind, placebo-controlled trial (LateTIME) of the National Heart, Lung, and Blood Institute-sponsored Cardiovascular Cell Therapy Research Network of 87 patients with significant LV dysfunction (LV ejection fraction [LVEF] ≤45%) following successful primary percutaneous coronary intervention (PCI) between July 8, 2008, and February 28, 2011. INTERVENTIONS Intracoronary infusion of 150 × 10(6) autologous BMCs (total nucleated cells) or placebo (BMC:placebo, 2:1) was performed within 12 hours of bone marrow aspiration after local automated cell processing. MAIN OUTCOME MEASURES Changes in global (LVEF) and regional (wall motion) LV function in the infarct and border zone between baseline and 6 months, measured by cardiac magnetic resonance imaging. Secondary end points included changes in LV volumes and infarct size. RESULTS A total of 87 patients were randomized (mean [SD] age, 57 [11] years; 83% men). Harvesting, processing, and intracoronary delivery of BMCs in this setting was feasible. Change between baseline and 6 months in the BMC group vs placebo for mean LVEF (48.7% to 49.2% vs 45.3% to 48.8%; between-group mean difference, -3.00; 95% CI, -7.05 to 0.95), wall motion in the infarct zone (6.2 to 6.5 mm vs 4.9 to 5.9 mm; between-group mean difference, -0.70; 95% CI, -2.78 to 1.34), and wall motion in the border zone (16.0 to 16.6 mm vs 16.1 to 19.3 mm; between-group mean difference, -2.60; 95% CI, -6.03 to 0.77) were not statistically significant. No significant change in LV volumes and infarct volumes was observed; both groups decreased by a similar amount at 6 months vs baseline. CONCLUSION Among patients with MI and LV dysfunction following reperfusion with PCI, intracoronary infusion of autologous BMCs vs intracoronary placebo infusion, 2 to 3 weeks after PCI, did not improve global or regional function at 6 months. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00684060.

Octamer binding proteins confer transcriptional activity in early mouse embryogenesis.

Oct4 and Oct5 are two mouse maternally expressed proteins binding to the octamer motif. Both are found in unfertilized oocytes and embryonic stem cells, whereas Oct4 is also found in primordial germ cells. In this study, the activity of the octamer motif was analysed in two embryonic stem cell lines containing Oct4 and Oct5, the teratocarcinoma‐derived cell line F9 and the blastocyst‐derived cell line D3. It is known that oligomerization of the octamer motif creates a powerful B‐cell specific enhancer. As shown here, this oligomerized transcriptional element is also a very strong enhancer in F9 and D3 embryonic stem cells. After differentiation of the stem cells, both enhancer activity and the amount of the octamer binding proteins decrease. An intact octamer stimulates heterologous promoters in embryonic stem cells, whereas mutations in the octamer motif abolish transcriptional stimulation and binding of the octamer factors. The use of transgenic embryos demonstrates transcriptional activation in the inner cell mass but not in the trophoblast of blastocysts. The results indicate that Oct4 and Oct5 are active early in mouse development.

Cytoplasmic Thioredoxin Reductase Is Essential for Embryogenesis but Dispensable for Cardiac Development

ABSTRACT Two distinct thioredoxin/thioredoxin reductase systems are present in the cytosol and the mitochondria of mammalian cells. Thioredoxins (Txn), the main substrates of thioredoxin reductases (Txnrd), are involved in numerous physiological processes, including cell-cell communication, redox metabolism, proliferation, and apoptosis. To investigate the individual contribution of mitochondrial (Txnrd2) and cytoplasmic (Txnrd1) thioredoxin reductases in vivo, we generated a mouse strain with a conditionally targeted deletion of Txnrd1. We show here that the ubiquitous Cre-mediated inactivation of Txnrd1 leads to early embryonic lethality. Homozygous mutant embryos display severe growth retardation and fail to turn. In accordance with the observed growth impairment in vivo, Txnrd1-deficient embryonic fibroblasts do not proliferate in vitro. In contrast, ex vivo-cultured embryonic Txnrd1-deficient cardiomyocytes are not affected, and mice with a heart-specific inactivation of Txnrd1 develop normally and appear healthy. Our results indicate that Txnrd1 plays an essential role during embryogenesis in most developing tissues except the heart.

Multistep Nature of Microvascular Recruitment of Ex Vivo-expanded Embryonic Endothelial Progenitor Cells during Tumor Angiogenesis

Tissue neovascularization involves recruitment of circulating endothelial progenitor cells that originate in the bone marrow. Here, we show that a class of embryonic endothelial progenitor cells (Tie-2+, c-Kit+, Sca-1+, and Flk-1−/low), which were isolated at E7.5 of mouse development at the onset of vasculogenesis, retain their ability to contribute to tumor angiogenesis in the adult. Using intravital fluorescence videomicroscopy, we further defined the multistep process of embryonic endothelial progenitor cell (eEPC) homing and incorporation. Circulating eEPCs are specifically arrested in “hot spots” within the tumor microvasculature, extravasate into the interstitium, form multicellular clusters, and incorporate into functional vascular networks. Expression analysis and in vivo blocking experiments provide evidence that the initial cell arrest of eEPC homing is mediated by E- and P-selectin and P-selectin glycoprotein ligand 1. This paper provides the first in vivo insights into the mechanisms of endothelial progenitor cell recruitment and, thus, indicates novel ways to interfere with pathological neovascularization.

Dorsomorphin, a Selective Small Molecule Inhibitor of BMP Signaling, Promotes Cardiomyogenesis in Embryonic Stem Cells

Background Pluripotent embryonic stem (ES) cells, which have the capacity to give rise to all tissue types in the body, show great promise as a versatile source of cells for regenerative therapy. However, the basic mechanisms of lineage specification of pluripotent stem cells are largely unknown, and generating sufficient quantities of desired cell types remains a formidable challenge. Small molecules, particularly those that modulate key developmental pathways like the bone morphogenetic protein (BMP) signaling cascade, hold promise as tools to study in vitro lineage specification and to direct differentiation of stem cells toward particular cell types. Methodology/ Principal Findings We describe the use of dorsomorphin, a selective small molecule inhibitor of BMP signaling, to induce myocardial differentiation in mouse ES cells. Cardiac induction is very robust, increasing the yield of spontaneously beating cardiomyocytes by at least 20 fold. Dorsomorphin, unlike the endogenous BMP antagonist Noggin, robustly induces cardiomyogenesis when treatment is limited to the initial 24-hours of ES cell differentiation. Quantitative-PCR analyses of differentiating ES cells indicate that pharmacological inhibition of BMP signaling during the early critical stage promotes the development of the cardiomyocyte lineage, but reduces the differentiation of endothelial, smooth muscle, and hematopoietic cells. Conclusions/ Significance Administration of a selective small molecule BMP inhibitor during the initial stages of ES cell differentiation substantially promotes the differentiation of primitive pluripotent cells toward the cardiomyocytic lineage, apparently at the expense of other mesodermal lineages. Small molecule modulators of developmental pathways like dorsomorphin could become versatile pharmacological tools for stem cell research and regenerative medicine.

Adherent Platelets Recruit and Induce Differentiation of Murine Embryonic Endothelial Progenitor Cells to Mature Endothelial Cells In Vitro

The homing and differentiation mechanisms of endothelial progenitor cells (EPCs) at sites of vascular lesions are unclear. To investigate whether platelets play a role in the recruitment and differentiation of EPCs, we made use of a robust mouse embryonic EPC (eEPC) line that reliably differentiates to a mature endothelial phenotype. We found that platelets stimulate chemotaxis and migration of these murine eEPCs. Further, the substantial adhesion of murine eEPCs on immobilized platelets that occurs under dynamic flow conditions is inhibited by neutralizing anti–P-selectin glycoprotein ligand-1 and anti–VLA-4 (β1-integrin) monoclonal antibodies but not by anti-CD11b (aM-integrin; macrophage antigen-1). Coincubation of murine eEPCs with platelets for 5 days induced differentiation of EPCs to mature endothelial cells as verified by positive von Willebrand factor immunofluorescence and detection of Weibel Palade bodies through electron microscopy. We conclude that platelets may play a critical part in the capture and subsequent differentiation of murine eEPCs at sites of vascular lesions, revealing a possible new role of platelets in neoendothelization after vascular injury.

Experimental myocardial infarction triggers canonical Wnt signaling and endothelial-to-mesenchymal transition

SUMMARY Despite available therapies, myocardial infarction (MI) remains a leading cause of death worldwide. Better understanding of the molecular and cellular mechanisms that regulate cardiac repair should help to improve the clinical outcome of MI patients. Using the reporter mouse line TOPGAL, we show that canonical (β-catenin-dependent) Wnt signaling is induced 4 days after experimental MI in subepicardial endothelial cells and perivascular smooth muscle actin (SMA)-positive (SMA+) cells. At 1 week after ischemic injury, a large number of canonical-Wnt-positive cells accumulated in the infarct area during granulation tissue formation. Coincidently with canonical Wnt activation, endothelial-to-mesenchymal transition (EndMT) was also triggered after MI. Using cell lineage tracing, we show that a significant portion of the canonical-Wnt-marked SMA+ mesenchymal cells is derived from endothelial cells. Canonical Wnt signaling induces mesenchymal characteristics in cultured endothelial cells, suggesting a direct role in EndMT. In conclusion, our study demonstrates that canonical Wnt activation and EndMT are molecular and cellular responses to MI and that canonical Wnt signaling activity is a characteristic property of EndMT-derived mesenchymal cells that take part in cardiac tissue repair after MI. These findings could lead to new strategies to improve the course of cardiac repair by temporal and cell-type-specific manipulation of canonical Wnt signaling.