Shaw-Fang Yet

Heme oxygenase-1 promotes neovascularization in ischemic heart by coinduction of VEGF and SDF-1

Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with multiple protective functions in cardiovascular systems. Studies have shown that the timely cardiac HO-1 overexpression at acute phase of ischemic infarction (MI) provides protection via its anti-apoptotic and anti-inflammatory effects. Here we demonstrate that a delayed HO-1 transduction mediated by a recombinant adeno-associated virus in ischemic hearts of mice with permanent coronary artery ligation significantly attenuated left ventricular fibrosis and cardiac dysfunctions examined at 4 weeks post MI. HO-1-mediated protection was correlated with enhanced vascularization in the ischemic myocardium. HO-1 gene transfer resulted in a notable increase in the number of c-kit(+)- stem cells recruited to the infarcted area at 10 days after ligation. HO-1-mediated stem cell recruitment was also demonstrated in the heart of non-ischemic mice receiving intravenous infusion of green fluorescent protein-bearing bone marrow stem cells. Additional experiments revealed that vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) were highly induced in HO-1 transduced myocardium. Mononuclear cell infiltration was evident and colocalized with angiogenic factors in the same region. Flow cytometry analysis of the mononuclear cells isolated from HO-1-transduced left ventricles revealed that over 50% of cells expressed CD34, a marker of hematopoietic stem cells and endothelial progenitor cells. VEGF and SDF-1 blockade by neutralizing antibodies significantly attenuated HO-1-mediated neovascularization and protection in infarcted mice. These data suggest that cardiac HO-1 gene transfer post MI provides protection at least in part by promoting neovascularization through inducing angiogenic factors and the recruitment of circulating progenitor/stem cells.

After vascular injury, heme oxygenase-1/carbon monoxide enhances re-endothelialization via promoting mobilization of circulating endothelial progenitor cells

Summary.u2002 Backgound: Heme oxygenase‐1 (HO‐1), a heme degradation enzyme with multiple vasoprotective functions, is systemically induced in pathophysiological states associated with oxidative stress. Objectives: To evaluate the impact of systemic HO‐1 expression on circulating endothelial progenitor cells (EPCs) and re‐endothelialization after vascular injury in an animal model. Methods: Mice received an intravenous (i.v.) injection of the adenovirus‐bearing HO‐1 gene (Adv‐HO‐1). The serum levels of vascular endothelial growth factor (VEGF) and stromal cell‐derived factor‐1 (SDF‐1) were determined by ELISA and gene expression examined by quantitative real‐time PCR. Circulating EPCs were characterized by flow cytometry and in vitro culture. EPC recruitment and re‐endothelialization in injured arteries were assessed in mice receiving GFP+‐bone marrow transplantation and guide wire‐induced carotid injury. The effect of carbon monoxide (CO), a byproduct from heme degradation by HO‐1, was assessed by exposing mice to 250u2003p.p.m. CO for 2u2003hu2003day−1. Results: Systemic HO‐1 induction led to elevated serum levels of VEGF and SDF‐1 and an increase in circulating EPCs. The re‐endothelialization of denuded vessels was accelerated in mice with systemic HO‐1 overexpression. A further experiment demonstrated that both EPC mobilization and re‐endothelialization were significantly attenuated in mice with HO‐1 deficiency. The increase in EPC mobilization and enhanced re‐endothelialization was also observed in mice exposed to CO prior to carotid injury. The CO‐mediated effect was associated with an increase in circulating SDF‐1 but not VEGF. Conclusion: These findings support a vital role of HO‐1 and its reaction byproduct, CO, in vascular repair through enhancing EPC mobilization.

Current applications of human pluripotent stem cells: Possibilities and challenges

Stem cells are self-renewable cells with the differentiation capacity to develop into somatic cells with biological functions. This ability to sustain a renewable source of multi- and/or pluripotential differentiation has brought new hope to the field of regenerative medicine in terms of cell therapy and tissue engineering. Moreover, stem cells are invaluable tools as in vitro models for studying diverse fields, from basic scientific questions such as developmental processes and lineage commitment, to practical application including drug screening and testing. The stem cells with widest differentiation potential are pluripotent stem cells (PSCs), which are rare cells with the ability to generate somatic cells from all three germ layers. PSCs are considered the most optimal choice for therapeutic potential of stem cells, bringing new impetus to the field of regenerative medicine. In this article, we discuss the therapeutic potential of human PSCs (hPSCs) including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), reviewing the current preclinical and clinical data using these stem cells. We describe the classification of different sources of hPSCs, ongoing research, and currently encountered clinical obstacles of these novel and versatile human stem cells.

Endogenous KLF4 Expression in Human Fetal Endothelial Cells Allows for Reprogramming to Pluripotency With Just OCT3/4 and SOX2—Brief Report

Objective—The introduction of 4 transcription factors—c-MYC, OCT3/4, SOX2, and KLF4—can reprogram somatic cells back to pluripotency. However, some of the factors used are oncogenic, making therapeutic application unfeasible. Although the use of adult stem cells expressing high endogenous levels of some of these factors allows for reprogramming with fewer exogenous genes, such cells are rare and may have accumulated genetic mutations. Our goal was to reprogram human somatic cells without oncogenic factors. We found that high endogenous expression of KLF4 in human umbilical vein endothelial cells (HUVECs) allows for generation of induced pluripotent stem cells (iPSCs) with just 2 nononcogenic factors, OCT3/4 and SOX2. Methods and Results—HUVECs were infected with lentivirus containing OCT4 and SOX2 for generation of iPSCs. These 2-factor HUVEC iPSCs were morphologically similar to embryonic stem cells, express endogenous pluripotency markers postreprogramming, and can differentiate toward lineages of all 3 germ layers both in vitro and in vivo. Conclusion—iPSCs can be generated from HUVECs with only 2 nononcogenic factors. The use of fetal cells for reprogramming without oncogenic factors may provide an efficient in vitro model for human iPSC research, as well as a novel source for possible therapeutic use.

Myeloid Heme Oxygenase-1 Haploinsufficiency Reduces High Fat Diet-Induced Insulin Resistance by Affecting Adipose Macrophage Infiltration in Mice

Increased adipose tissue macrophages contribute to obesity-induced metabolic syndrome. Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with potent anti-inflammatory and proangiogenic activities in macrophages. However, the role of macrophage HO-1 on obesity-induced adipose inflammation and metabolic syndrome remains unclear. Here we show that high-fat diet (HFD) feeding in C57BL/6J mice induced HO-1 expression in the visceral adipose tissue, particularly the stromal vascular fraction. When the irradiated C57BL/6J mice reconstituted with wild-type or HO-1+/− bone marrow were fed with HFD for over 24 weeks, the HO-1+/− chimeras were protected from HFD-induced insulin resistance and this was associated with reduced adipose macrophage infiltration and angiogenesis, suggesting that HO-1 affects myeloid cell migration toward adipose tissue during obesity. In vivo and in vitro migration assays revealed that HO-1+/− macrophages exhibited an impaired migration response. Chemoattractant-induced phosphorylation of p38 and focal adhesion kinase (FAK) declined faster in HO-1+/− macrophages. Further experiments demonstrated that carbon monoxide and bilirubin, the byproducts derived from heme degradation by HO-1, enhanced macrophage migration by increasing phosphorylation of p38 and FAK, respectively. These data disclose a novel role of hematopoietic cell HO-1 in promoting adipose macrophage infiltration and the development of insulin resistance during obesity.

Exacerbation of Oxidative Stress-Induced Cell Death and Differentiation in Induced Pluripotent Stem Cells Lacking Heme Oxygenase-1

Embryonic stem cells (ESCs) are promising donor sources in cell therapies for various diseases. Although low levels of reactive oxygen species (ROS) are necessary for the maintenance of stem cells, increased ROS levels initiate differentiation and cell damage. We and others have previously demonstrated that heme oxygenase (HO)-1, a stress response protein with antioxidative and anti-inflammatory properties, plays critical protective functions in cardiovascular and other diseases. However, the functions of HO-1 in ESCs remain to be elucidated. Our goal was to investigate the roles of HO-1 in ESC survival and differentiation. Due to the lack of HO-1-deficient ESCs, we used Oct3/4, Sox2, c-Myc, and Klf4 retroviruses to reprogram mouse embryonic fibroblasts into induced pluripotent stem (iPS) cells of different HO-1 genotypes. These iPS-HO-1 cells exhibited characteristics of mouse ESCs (mESCs) and formed teratomas that were composed of cell types of all 3 germ layers after injected into severe combined immunodeficiency mice. In response to oxidant stress, iPS-HO-1(-/-) cells accumulated higher levels of intracellular ROS compared with D3 mESCs or iPS-HO-1(+/+) cells and were more prone to oxidant-induced cell death. Spontaneous differentiation experiments revealed that Oct4 levels were significantly lower in iPS-HO-1(-/-) cells after leukemia inhibitory factor withdrawal and removal of feeders. Further, during the course of spontaneous differentiation, iPS-HO-1(-/-) cells had enhanced Erk1/2 phosphorylation, which has been linked to ESC differentiation. By the loss-of-function approach using iPS-HO-1(-/-) cells, our results demonstrate that a lack of HO-1 renders iPS cells more prone to oxidative stress-induced cell death and differentiation.

A novel TFG mutation causes Charcot-Marie-Tooth disease type 2 and impairs TFG function

Objective: To describe a novel mutation in TRK-fused gene (TFG) as a new cause of dominant axonal Charcot-Marie-Tooth disease (CMT) identified by exome sequencing and further characterized by in vitro functional studies. Methods: Exome sequencing and linkage analysis were utilized to investigate a large Taiwanese family with a dominantly inherited adult-onset motor and sensory axonal neuropathy in which mutations in common CMT2-implicated genes had been previously excluded. Functional effects of the mutant gene products were investigated in vitro. Results: Exome sequencing of 2 affected individuals in this family revealed a novel heterozygous mutation, c.806G>T (p.Gly269Val), in TFG that perfectly cosegregates with the CMT2 phenotype in all 27 family members. This mutation occurs at an evolutionarily conserved residue and is absent in the 1,140 ethnically matched control chromosomes. Genome-wide linkage study also supported its disease-causative role. Cell transfection studies showed that the TFG p.Gly269Val mutation increased the propensity of TFG proteins to form aggregates, resulting in sequestration of both mutant and wild-type TFG proteins and might thus deplete functional TFG molecules. The secreted Gaussia luciferase reporter assay demonstrated that inhibition of endogenous TFG compromised the protein secretion pathways, which could only be rescued by expressing wild-type TFG but not the p.Gly269Val altered proteins. TFG mutation was not found in 55 additional unrelated patients with CMT2, suggesting its rarity. Conclusion: This study identifies a new cause of dominant CMT2 and highlights the importance of TFG in the protein secretory pathways that are essential for proper functioning of the human peripheral nervous system.

Endothelium-Derived 5-Methoxytryptophan Is a Circulating Anti-inflammatory Molecule that Blocks Systemic Inflammation

RATIONALEnSystemic inflammation has emerged as a key pathophysiological process that induces multiorgan injury and causes serious human diseases. Endothelium is critical in maintaining cellular and inflammatory homeostasis, controlling systemic inflammation, and progression of inflammatory diseases. We postulated that endothelium produces and releases endogenous soluble factors to modulate inflammatory responses and protect against systemic inflammation.nnnOBJECTIVEnTo identify endothelial cell-released soluble factors that protect against endothelial barrier dysfunction and systemic inflammation.nnnMETHODS AND RESULTSnWe found that conditioned medium of endothelial cells inhibited cyclooxgenase-2 and interleukin-6 expression in macrophages stimulated with lipopolysaccharide. Analysis of conditioned medium extracts by liquid chromatography-mass spectrometry showed the presence of 5-methoxytryptophan (5-MTP), but not other related tryptophan metabolites. Furthermore, endothelial cell-derived 5-MTP suppressed lipopolysaccharide-induced inflammatory responses and signaling in macrophages and endotoxemic lung tissues. Lipopolysaccharide suppressed 5-MTP level in endothelial cell-conditioned medium and reduced serum 5-MTP level in the murine sepsis model. Intraperitoneal injection of 5-MTP restored serum 5-MTP accompanied by the inhibition of lipopolysaccharide-induced endothelial leakage and suppression of lipopolysaccharide- or cecal ligation and puncture-mediated proinflammatory mediators overexpression. 5-MTP administration rescued lungs from lipopolysaccharide-induced damages and prevented sepsis-related mortality. Importantly, compared with healthy subjects, serum 5-MTP level in septic patients was decreased by 65%, indicating an important clinical relevance.nnnCONCLUSIONSnWe conclude that 5-MTP belongs to a novel class of endothelium-derived protective molecules that defend against endothelial barrier dysfunction and excessive systemic inflammatory responses.

14-3-3σ regulates β-catenin-mediated mouse embryonic stem cell proliferation by sequestering GSK-3β.

Background Pluripotent embryonic stem cells are considered to be an unlimited cell source for tissue regeneration and cell-based therapy. Investigating the molecular mechanism underlying the regulation of embryonic stem cell expansion is thus important. 14-3-3 proteins are implicated in controlling cell division, signaling transduction and survival by interacting with various regulatory proteins. However, the function of 14-3-3 in embryonic stem cell proliferation remains unclear. Methodology and Principal Findings In this study, we show that all seven 14-3-3 isoforms were detected in mouse embryonic stem cells. Retinoid acid suppressed selectively the expression of 14-3-3σ isoform. Knockdown of 14-3-3σ with siRNA reduced embryonic stem cell proliferation, while only 14-3-3σ transfection increased cell growth and partially rescued retinoid acid-induced growth arrest. Since the growth-enhancing action of 14-3-3σ was abrogated by β-catenin knockdown, we investigated the influence of 14-3-3σ overexpression on β-catenin/GSK-3β. 14-3-3σ bound GSK-3β and increased GSK-3β phosphorylation in a PI-3K/Akt-dependent manner. It disrupted β-catenin binding by the multiprotein destruction complex. 14-3-3σ overexpression attenuated β-catenin phosphorylation and rescued the decline of β-catenin induced by retinoid acid. Furthermore, 14-3-3σ enhanced Wnt3a-induced β-catenin level and GSK-3β phosphorylation. DKK, an inhibitor of Wnt signaling, abolished Wnt3a-induced effect but did not interfere GSK-3β/14-3-3σ binding. Significance Our findings show for the first time that 14-3-3σ plays an important role in regulating mouse embryonic stem cell proliferation by binding and sequestering phosphorylated GSK-3β and enhancing Wnt-signaled GSK-3β inactivation. 14-3-3σ is a novel target for embryonic stem cell expansion.

Prostacyclin and PPARα Agonists Control Vascular Smooth Muscle Cell Apoptosis and Phenotypic Switch through Distinct 14-3-3 Isoforms

We hypothesized that prostacyclin (PGI2) protects vascular smooth muscle cell (VSMC) against apoptosis and phenotypic switch through peroxisome proliferator-activated receptor-α (PPARα) activation and 14-3-3 upregulation. Here we showed that transfection of rat aortic VSMC, A-10, with PGI2-producing vectors, Ad-COPI, resulted in attenuated H2O2-induced apoptosis accompanied by a selective increase in 14-3-3β and 14-3-3θ expression. Carbaprostacyclin (cPGI2) and Wy14,643 exerted a similar effect. The effects of PGI2 were abrogated by MK886, a PPARα antagonist, but not GSK3787, a PPARδ antagonist. PPARα transfection upregulated 14-3-3β and θ expression and attenuated H2O2-induced apoptosis. H2O2-induced 14-3-3β but not 14-3-3θ degradation was blocked by a caspase 3 inhibitor. Furthermore, 14-3-3β but not 14-3-3θ overexpression reduced, while 14-3-3β siRNA aggravated apoptosis. VSMC contractile proteins and serum response factor (SRF) were reduced in H2O2-treated A-10 cells which were concurrently prevented by caspase 3 inhibitor. By contrast, PGI2 prevented H2O2-induced SM22α and Calponin-1 degradation without influencing SRF. cPGI2 and Wy14,643 also effectively blocked VSMC phenotypic switch induced by growth factors (GFs). GFs suppressed 14-3-3β, θ, ε and η isoforms and cPGI2 prevented the decline of β, θ and η, but not ε. 14-3-3θ siRNA abrogated the protective effect of cPGI2 on SM22α and Calponin-1 while 14-3-3 θ or 14-3-3β overexpression partially restored SM22α. These results indicated that PGI2 protects VSMCs via PPARα by upregulating 14-3-3β and 14-3-3θ. 14-3-3β upregulation confers resistance to apoptosis whereas 14-3-3θ and β upregulation protects SM22α and Calponin-1 from degradation.