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ABSTRACT: Loss-of-function mutation of Jup has been associated with Naxos disease, which is characterized by arrhythmogenic cardiomyopathy and the cutaneous disorder palmoplantar keratoderma. Previously, we have shown that genetic ablation of Jup in cardiomyocytes in mice leads to arrhythmogenic cardiomyopathy similar to Naxos disease in humans. Currently, to determine the pathogenesis of Naxos disease-associated keratoderma, we generated Jup mutant mice by inactivating Jup restrictively in keratinocytes. Jup mutant mice largely recapitulated the clinical features of human palmoplantar keratoderma: overcornification and thickening of the epidermis. Jup mutant mice also suffered skin ulceration and inflammation. Cell apoptosis and proliferation were significantly elevated in Jup mutant epidermis. Ultrastructural analyses revealed the disruption of the assembly of desmosomes and adherens junctions in Jup mutant epidermis. We also demonstrated the compensational increase in β-catenin at Jup mutant cell-cell junctions without altering its signaling activities. Our findings provide important insights for understanding the pathogenesis of human palmoplantar keratoderma.
Journal of Biological Chemistry 02/2012; 287(13):10435-43. · 4.77 Impact Factor
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ABSTRACT: FA is a genetic disorder characterized by BM failure, developmental defects, and cancer predisposition. Previous studies suggest that FA patients exhibit alterations in immunologic function. However, it is unclear whether the defects are immune cell-autonomous or secondary to leukopenia from evolving BM failure. Given the central role that macrophages have in the innate immune response, inflammation resolution, and antigen presentation for acquired immunity, we examined whether macrophages from Fancc-/- mice exhibit impaired function. Peritoneal inflammation induced by LPS or sodium periodate resulted in reduced monocyte/macrophage recruitment in Fancc-/- mice compared with WT controls. Fancc-/- mice also had decreased inflammatory monocytes mobilized into the peripheral blood after LPS treatment compared with controls. Furthermore, Fancc-/- peritoneal macrophages displayed cell-autonomous defects in function, including impaired adhesion to FN or endothelial cells, reduced chemoattractant-mediated migration, and decreased phagocytosis. Moreover, dysregulated F-actin rearrangement was detected in Fancc-/- macrophages after adhesion to FN, which was consistent with an observed reduction in RhoA-GTP levels. Importantly, these data suggest that impaired cytoskeletal rearrangements in Fancc-/- macrophages may be the common mechanism responsible for cell-autonomous defects detected in vitro, as well as altered monocyte/macrophage trafficking in vivo.
Journal of leukocyte biology 11/2011; 91(2):333-40. · 4.99 Impact Factor
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Deqiang Li,
Ying Liu,
Mitsunori Maruyama,
Wuqiang Zhu,
Hanying Chen,
Wenjun Zhang,
Sean Reuter,
Shien-Fong Lin, Laura S Haneline,
Loren J Field,
Peng-Sheng Chen,
Weinian Shou
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ABSTRACT: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inheritable myocardial disorder associated with fibrofatty replacement of myocardium and ventricular arrhythmia. A subset of ARVC is categorized as Naxos disease, which is characterized by ARVC and a cutaneous disorder. A homozygous loss-of-function mutation of the Plakoglobin (Jup) gene, which encodes a major component of the desmosome and the adherens junction, had been identified in Naxos patients, although the underlying mechanism remained elusive. We generated Jup mutant mice by ablating Jup in cardiomyocytes. Jup mutant mice largely recapitulated the clinical manifestation of human ARVC: ventricular dilation and aneurysm, cardiac fibrosis, cardiac dysfunction and spontaneous ventricular arrhythmias. Ultra-structural analyses revealed that desmosomes were absent in Jup mutant myocardia, whereas adherens junctions and gap junctions were preserved. We found that ventricular arrhythmias were associated with progressive cardiomyopathy and fibrosis in Jup mutant hearts. Massive cell death contributed to the cardiomyocyte dropout in Jup mutant hearts. Despite the increase of β-catenin at adherens junctions in Jup mutant cardiomyoicytes, the Wnt/β-catenin-mediated signaling was not altered. Transforming growth factor-beta-mediated signaling was found significantly elevated in Jup mutant cardiomyocytes at the early stage of cardiomyopathy, suggesting an important pathogenic pathway for Jup-related ARVC. These findings have provided further insights for the pathogenesis of ARVC and potential therapeutic interventions.
Human Molecular Genetics 08/2011; 20(23):4582-96. · 7.64 Impact Factor
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ABSTRACT: FANCD2, a key factor in the FANC-BRCA1 pathway is monoubiquitinated and targeted to discrete nuclear foci following DNA damage. Since monoubiquitination of FANCD2 is a crucial indicator for cellular response to DNA damage, we monitored the fate of FANCD2 and its monoubiquitination following DNA damage. Disappearance of FANCD2 protein was induced following DNA damage in a dose-dependent manner, which correlated with degradation of BRCA1 and poly-ADP ribose polymerase (PARP), known targets for caspase-mediated apoptosis. Disappearance of FANCD2 was not affected by a proteasome inhibitor but was blocked by a caspase inhibitor. DNA damage-induced disappearance of FANCD2 was also observed in cells lacking FANCA, suggesting that disappearance of FANCD2 does not depend on FANC-BRCA1 pathway and FANCD2 monoubiquitination. In keeping with this, cells treated with TNF-α, an apoptotic stimulus without causing any DNA damage, also induced disappearance of FANCD2 without monoubiquitination. Together, our data suggest that FANCD2 is a target for caspase-mediated apoptotic pathway, which may be an early indicator for apoptotic cell death.
Journal of Cellular Biochemistry 04/2011; 112(9):2383-91. · 2.87 Impact Factor
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Deqiang Li,
Mark A Hallett,
Wuqiang Zhu,
Michael Rubart,
Ying Liu,
Zhenyun Yang,
Hanying Chen, Laura S Haneline,
Rebecca J Chan,
Robert J Schwartz,
Loren J Field,
Simon J Atkinson,
Weinian Shou
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ABSTRACT: Dishevelled-associated activator of morphogenesis 1 (Daam1), a member of the formin protein family, plays an important role in regulating the actin cytoskeleton via mediation of linear actin assembly. Previous functional studies of Daam1 in lower species suggest its essential role in Drosophila trachea formation and Xenopus gastrulation. However, its in vivo physiological function in mammalian systems is largely unknown. We have generated Daam1-deficient mice via gene-trap technology and found that Daam1 is highly expressed in developing murine organs, including the heart. Daam1-deficient mice exhibit embryonic and neonatal lethality and suffer multiple cardiac defects, including ventricular noncompaction, double outlet right ventricles and ventricular septal defects. In vivo genetic rescue experiments further confirm that the lethality of Daam1-deficient mice results from the inherent cardiac abnormalities. In-depth analyses have revealed that Daam1 is important for regulating filamentous actin assembly and organization, and consequently for cytoskeletal function in cardiomyocytes, which contributes to proper heart morphogenesis. Daam1 is also found to be important for proper cytoskeletal architecture and functionalities in embryonic fibroblasts. Biochemical analyses indicate that Daam1 does not regulate cytoskeletal organization through RhoA, Rac1 or Cdc42. Our study highlights a crucial role for Daam1 in regulating the actin cytoskeleton and tissue morphogenesis.
Development 01/2011; 138(2):303-15. · 6.60 Impact Factor
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ABSTRACT: The purpose of this study was to examine whether women with gestational diabetes mellitus (GDM) and their offspring have reduced endothelial progenitor cell subsets and vascular reactivity.
Women with GDM, healthy control subjects, and their infants participated. Maternal blood and cord blood were assessed for colony-forming unit-endothelial cells and endothelial progenitor cell subsets with the use of polychromatic flow cytometry. Cord blood endothelial colony-forming cells were enumerated. Vascular reactivity was tested by laser Doppler imaging.
Women with GDM had fewer CD34, CD133, CD45, and CD31 cells (circulating progenitor cells [CPCs]) at 24-32 weeks' gestation and 1-2 days after delivery, compared with control subjects. No differences were detected in colony-forming unit-endothelial cells or colony-forming unit-endothelial cells. In control subjects, CPCs were higher in the third trimester, compared with the postpartum period. Cord blood from GDM pregnancies had reduced CPCs. Vascular reactivity was not different between GDM and control subjects.
The normal physiologic increase in CPCs during pregnancy is impaired in women with GDM, which may contribute to endothelial dysfunction and GDM-associated morbidities.
American journal of obstetrics and gynecology 12/2010; 204(3):254.e8-254.e15. · 3.28 Impact Factor
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ABSTRACT: To evaluate whether counts of circulating colony forming unit-endothelial cells (CFU-ECs), cells co-expressing CD34, CD133, and CD31 (CD34+CD133+CD31+), and CD34+CD45- cells are altered in adolescents with type 1 diabetes and if the changes in counts correlate with endothelial dysfunction.
Adolescents with diabetes (ages 18 to 22 years) and race- and sex-matched control subjects were studied. We assessed circulating CFU-ECs, using colony assays, and CD34+CD133+CD31+ and CD34+CD45- cells, using poly-chromatic flow cytometry. CFU-ECs and CD34+CD133+CD31+ are hematopoietic-derived progenitors that inversely correlate with cardiovascular risk in adults. CD34+CD45- cells are enriched for endothelial cells with robust vasculogenic potential. Vascular reactivity was tested by laser Doppler iontophoresis.
Subjects with diabetes had lower CD34+CD133+CD31+ cells, a trend toward reduced CFU-ECs, and increased CD34+CD45- cells compared with control subjects. Endothelium-dependent vasodilation was impaired in subjects with diabetes, which correlated with reductions in circulating CD34+CD133+CD31+ cells.
Long-term sequelae of type 1 diabetes include vasculopathies. Endothelial progenitor cells promote vascular health by facilitating endothelial integrity and function. Lower CD34+CD133+CD31+ cells may be a harbinger of future macrovascular disease risk. Higher circulating CD34+CD45- cells may reflect ongoing endothelial damage. These cells are potential biomarkers to guide therapeutic interventions to enhance endothelial function and to prevent progression to overt vascular disease.
The Journal of pediatrics 10/2010; 157(4):540-6. · 4.02 Impact Factor
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Matthew R Distasi,
Jamie Case,
Matthew A Ziegler,
Mary C Dinauer,
Mervin C Yoder, Laura S Haneline,
Michael C Dalsing,
Steven J Miller,
Carlos A Labarrere,
Michael P Murphy,
David A Ingram,
Joseph L Unthank
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ABSTRACT: While tissue perfusion and angiogenesis subsequent to acute femoral artery occlusion are suppressed in NADPH oxidase 2 (Nox2)-null (Nox2(-/-)) mice, studies have not established the role of Nox2 in collateral artery enlargement. Rac2 is a small GTPase that binds Nox2 and activates Nox2-based NAD(P)H oxidase but, unlike Nox2, is primarily restricted to bone marrow-derived cells. In this study, we used Rac2-null (Rac2(-/-)) and Nox2(-/-) mice with a novel method of identifying primary hindlimb collaterals to investigate the hypothesis that collateral growth requires these molecules. When initial experiments performed with femoral ligation demonstrated similar perfusion and collateral growth in Rac2(-/-) and wild-type C57BL/6J (BL6) mice, subsequent experiments were performed with a more severe ischemia model, femoral artery excision. After femoral excision, tissue perfusion was suppressed in Rac2(-/-) mice relative to BL6 mice. Histological assessment of ischemic injury including necrotic and regenerated muscle fibers and lipid and collagen deposition demonstrated greater injury in Rac2(-/-) mice. The diameters of primary collaterals identified during Microfil injection with intravital microscopy were enlarged to a similar extent in BL6 and Rac2(-/-) mice. Intimal cells in collateral cross sections were increased in number in both strains and were CD31 positive and CD45 negative. Circulating leukocytes and CD11b(+) cells were increased more in Rac2(-/-) than BL6 animals. Experiments performed in Nox2(-/-) mice to verify that the unexpected results related to collateral growth were not unique to Rac2(-/-) mice gave equivalent results. The data demonstrate that, subsequent to acute femoral artery excision, perfusion recovery is impaired in Rac2(-/-) and Nox2(-/-) mice but that collateral luminal expansion and intimal cell recruitment/proliferation are normal. These novel results indicate that collateral luminal expansion and intimal cell recruitment/proliferation are not mediated by Rac2 and Nox2.
AJP Heart and Circulatory Physiology 02/2009; 296(3):H877-86. · 3.71 Impact Factor
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ABSTRACT: The underlying molecular mechanisms that promote bone marrow failure in Fanconi anemia are incompletely understood. Evidence suggests that enhanced apoptosis of hematopoietic precursors is a major contributing factor. Previously, enhanced apoptosis of Fanconi anemia type C-deficient (Fancc(-/-)) progenitors was shown to involve aberrant p38 MAPK activation. Given the importance of c-Jun N-terminal kinase (JNK) in the stress response, we tested whether enhanced apoptosis of Fancc(-/-) cells also involved altered JNK activation. In Fancc(-/-) murine embryonic fibroblasts, tumor necrosis factor alpha (TNF-alpha) induced elevated JNK activity. In addition, JNK inhibition protected Fancc(-/-) murine embryonic fibroblasts and c-kit(+) bone marrow cells from TNF-alpha-induced apoptosis. Importantly, hematopoietic progenitor assays demonstrated that JNK inhibition enhanced Fancc(-/-) colony formation in the presence of TNF-alpha. Competitive repopulation assays showed that Fancc(-/-) donor cells cultured with the JNK inhibitor had equivalent levels of donor chimerism compared with Fancc(-/-) donor cells cultured with vehicle control. In contrast, culturing Fancc(-/-) cells with a p38 MAPK inhibitor significantly increased repopulating ability, supporting an integral role of p38 MAPK in maintaining Fancc(-/-) hematopoietic stem cell function. Taken together, these data suggest that p38 MAPK, but not JNK, has a critical role in maintaining the engraftment of Fancc(-/-)-reconstituting cells under conditions of stress.
Blood 02/2009; 113(12):2655-60. · 9.90 Impact Factor
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Laura S Haneline
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ABSTRACT: The field of stem and progenitor cell biology is expanding. Much of the enthusiasm is based on the potential of using stem and progenitor cells as a cellular therapy for the treatment of human disease. Although the concept of using human embryonic stem cells for therapeutic indications is intriguing, significant challenges face investigators pursuing research in this area. Therefore, renewed scientific energy is focusing on the molecular pathways that differentiate a pluripotent embryonic stem cell from more-committed tissue-specific cells. Molecular mechanisms that govern tissue-specific stem and progenitor cell function are also topics of intense investigation, given that altered function of these cells may promote a variety of human pathologies including aging, vascular disease, and cancer. Considerable progress has been made, but a clear identification of the molecular signatures of stem and progenitor cells remains elusive. A growing body of literature demonstrates that distinct functional characteristics of stem and progenitor cells are under redox regulation. In this Forum Issue, evidence for redox regulation of tissue-specific stem and progenitor cells involved in hematopoiesis and vasculogenesis/angiogenesis is presented.
Antioxidants & Redox Signaling 12/2008; 10(11):1849-52. · 8.20 Impact Factor
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ABSTRACT: Circulating endothelial progenitor cells (EPCs) in adult human peripheral blood were identified in 1997. Since their original identification, EPCs have been extensively studied as biomarkers to assess the risk of cardiovascular disease in human subjects and as a potential cell therapeutic for vascular regeneration. EPCs are exposed to oxidative stress during vascular injury as residents of blood vessel walls or as circulating cells homing to sites of neovascularization. Given the links between oxidative injury, endothelial cell dysfunction, and vascular disease, recent investigation has focused on the responses of EPCs to oxidant stress and the molecular mechanisms that control redox regulation in these specialized cells. In this review, we discuss the various cell and flow-cytometric techniques used to define and isolate EPCs from circulating blood and the current human and mouse genetic data, which offer insights into redox control in EPC biology and angiogenesis. Finally, we review how EPC responses to oxidant stress may be a critical determinant in maintaining the integrity and function of the cardiovascular system and how perturbations of redox control in EPCs may lead to various human diseases.
Antioxidants & Redox Signaling 11/2008; 10(11):1895-907. · 8.20 Impact Factor
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ABSTRACT: Emerging data demonstrate that maternal diabetes has long-term health consequences for offspring, including the development of hypertension. In adults, circulating endothelial progenitor cells (EPCs) participate in vascular repair, and EPC numbers and function inversely correlate with the risk of developing vascular disease. Therefore, our objectives were to determine whether hyperglycemia or exposure to a diabetic intrauterine environment alters EPC function.
We used well-established clonogenic endothelial colony-forming cell (ECFC) assays and murine transplantation experiments to examine human vasculogenesis.
Both in vitro hyperglycemia and a diabetic intrauterine environment reduced ECFC colony formation, self-renewal capacity, and capillary-like tube formation in matrigel. This cellular phenotype was linked to premature senescence and reduced proliferation. Further, cord blood ECFCs from diabetic pregnancies formed fewer chimeric vessels de novo after transplantation into immunodeficient mice compared with neonatal ECFCs harvested from uncomplicated pregnancies. CONCLUSIONS; Collectively, these data demonstrate that hyperglycemia or exposure to a diabetic intrauterine environment diminishes neonatal ECFC function both in vitro and in vivo, providing potential mechanistic insights into the long-term cardiovascular complications observed in newborns of diabetic pregnancies.
Diabetes 04/2008; 57(3):724-31. · 8.29 Impact Factor
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ABSTRACT: Several hematopoietic stem-cell (HSC) regulators are controlled by ubiquitin-mediated proteolysis, so the ubiquitin pathway might modulate HSC function. However, this hypothesis has not been formally tested. Cul4A encodes a core subunit of one ubiquitin ligase. Whereas Cul4A-deficient embryos die in utero, Cul4A-haploinsufficient mice are viable but exhibit abnormal hematopoiesis (fewer erythroid and primitive myeloid progenitors). Given these data, we examined whether Cul4A(+/-) HSCs might also be impaired. Using bone marrow transplantation assays, we determined that Cul4A(+/-) HSCs exhibit defects in engraftment and self-renewal capacity. These studies are the first to demonstrate that ubiquitin-mediated protein degradation is important for HSC function. Further, they indicate that a Cul4A ubiquitin ligase targets for degradation one or multiple HSC regulators.
Blood 11/2007; 110(7):2704-7. · 9.90 Impact Factor
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ABSTRACT: Endothelial progenitor cells (EPCs) are used for angiogenic therapies or as biomarkers to assess cardiovascular disease risk. However, there is no uniform definition of an EPC, which confounds EPC studies. EPCs are widely described as cells that coexpress the cell-surface antigens CD34, AC133, and vascular endothelial growth factor receptor-2 (VEGFR-2). These antigens are also expressed on primitive hematopoietic progenitor cells (HPCs). Remarkably, despite their original identification, CD34+AC133+VEGFR-2+ cells have never been isolated and simultaneously plated in hematopoietic and endothelial cell (EC) clonogenic assays to assess the identity of their clonal progeny, which are presumably the cellular participants in vascular regeneration.
CD34+AC133+VEGFR-2+ cells were isolated from human umbilical cord blood (CB) or granulocyte colony-stimulating factor-mobilized peripheral blood and assayed for either EPCs or HPCs.
CD34+AC133+VEGFR-2+ cells did not form EPCs and were devoid of vessel forming activity. However, CD34+AC133+VEGFR-2+ cells formed HPCs and expressed the hematopoietic lineage-specific antigen, CD45. We next tested whether EPCs could be separated from HPCs by immunoselection for CD34 and CD45. CD34+CD45+ cells formed HPCs but not EPCs, while CD34+CD45- cells formed EPCs but not HPCs.
Therefore, CD34+AC133+VEGFR-2+ cells are HPCs that do not yield EC progeny, and the biological mechanism for their correlation with cardiovascular disease needs to be reexamined.
Experimental Hematology 08/2007; 35(7):1109-18. · 2.90 Impact Factor
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David A Ingram,
Theresa R Krier,
Laura E Mead,
Colleen McGuire,
Daniel N Prater,
Janak Bhavsar,
M Reza Saadatzadeh,
Khadijeh Bijangi-Vishehsaraei,
Fang Li,
Mervin C Yoder, Laura S Haneline
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ABSTRACT: Endothelial progenitor cells (EPCs) circulate in the peripheral blood and reside in blood vessel walls. A hierarchy of EPCs exists where progenitors can be discriminated based on their clonogenic potential. EPCs are exposed to oxidative stress during vascular injury as residents of blood vessel walls or as circulating cells homing to sites of neovascularization. Given the links between oxidative injury, endothelial cell dysfunction, and vascular disease, we tested whether EPCs were sensitive to oxidative stress using newly developed clonogenic assays. Strikingly, in contrast to previous reports, we demonstrate that the most proliferative EPCs (high proliferative potential-endothelial colony-forming cells and low proliferative potential-endothelial colony-forming cells) had decreased clonogenic capacity after oxidant treatment. In addition, EPCs exhibited increased apoptosis and diminished tube-forming ability in vitro and in vivo in response to oxidative stress, which was directly linked to activation of a redox-dependent stress-induced kinase pathway. Thus, this study provides novel insights into the effect of oxidative stress on EPCs. Furthermore, this report outlines a framework for understanding how oxidative injury leads to vascular disease and potentially limits the efficacy of transplantation of EPCs into ischemic tissues enriched for reactive oxygen species and oxidized metabolites.
Stem Cells 03/2007; 25(2):297-304. · 7.78 Impact Factor
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Yue Si,
Samantha Ciccone,
Feng-Chun Yang,
Jin Yuan,
Daisy Zeng,
Shi Chen,
Henri J van de Vrugt,
John Critser,
Fre Arwert, Laura S Haneline,
D Wade Clapp
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ABSTRACT: Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. However, strategies to enhance the mobilization, transduction, and engraftment of exogenous stem cells are required to optimize efficacy prior to widespread clinical use. Hypersensitivity of Fancc-/- cells to interferon-gamma (IFN-gamma), a nongenotoxic immune-regulatory cytokine, enhances engraftment of syngeneic wild-type (WT) cells in Fancc-/- mice. However, whether this phenotype is of broad relevance in other FA complementation groups is unresolved. Here we show that primitive and mature myeloid progenitors in Fanca-/- and Fancg-/- mice are hypersensitive to IFN-gamma and that in vivo infusion of IFN-gamma at clinically relevant concentrations was sufficient to allow consistent long-term engraftment of isogenic WT repopulating stem cells. Given that FANCA, FANCC, and FANCG complementation groups account for more than 90% of all FA patients, these data provide evidence that IFN-gamma conditioning may be a useful nongenotoxic strategy for myelopreparation in FA patients.
Blood 01/2007; 108(13):4283-7. · 9.90 Impact Factor
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ABSTRACT: Class I(A) phosphatidylinositol-3 kinase (PI-3K) is a lipid kinase, which is activated in blood cells by hematopoietic growth factors. In vitro experiments using chemical inhibitors of PI-3K suggest that this kinase is potentially important for hematopoietic stem and progenitor cell (HSC/P) function, and recent studies identify PI-3K as a therapeutic target in treating different leukemias and lymphomas. However, the role of PI-3K in regulating fetal liver or adult hematopoiesis in vivo is unknown. Therefore, we examined PI-3K-deficient embryos generated by a targeted deletion of the p85alpha and p85beta regulatory subunits of PI-3K (p85alpha-/-p85beta+/-). The absolute frequency and number of hematopoietic progenitor cells were reduced in p85alpha-/- p85beta+/- fetal livers compared with wild-type (WT) controls. Further, p85alpha-/-p85beta+/- fetal liver hematopoietic stem cells (HSCs) had decreased multilineage repopulating ability in vivo compared with WT controls in competitive repopulation assays. Finally, purified p85alpha-/-p85beta+/- c-kit+ cells had a decrease in proliferation in response to kit ligand (kitL), a growth factor important for controlling HSC function in vivo. Collectively, these data identify PI-3K as an important regulator of HSC function and potential therapeutic target in treating leukemic stem cells.
Blood 03/2006; 107(4):1375-82. · 9.90 Impact Factor
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ABSTRACT: Fanconi anemia (FA) is a chromosomal instability disorder characterized by progressive bone marrow failure. Experimental evidence suggests that enhanced oxidant and myelosuppressive cytokine-mediated apoptosis of hematopoietic stem and progenitor cells contributes to the pathogenesis of marrow failure in FA. However, the molecular mechanisms responsible for the apoptotic phenotype in hematopoietic cells are incompletely understood. Recent data in Fancc-/- murine embryonic fibroblasts (MEFs) implicate increased oxidant-induced apoptotic signaling through the redox-dependent protein, apoptosis signal-regulating kinase 1 (Ask1). Here, we examined whether altered Ask1 signaling participated in the proapoptotic phenotype of primary Fancc-/- MEFs and hematopoietic progenitors treated with the myelosuppressive cytokine tumor necrosis factor-alpha (TNF-alpha). Our data indicate that TNF-alpha induces hyperactivation of Ask1 and the downstream effector p38 in Fancc-/- MEFs. In addition,Ask1 inactivation in Fancc-/- MEFs and hematopoietic progenitors restored survival to wild-type (WT) levels in the presence of TNF-alpha. Furthermore, targeting the Ask1 pathway by using either antioxidants or a p38 inhibitor protected Fancc-/- MEFs and c-kit+ cells from TNF-alpha-induced apoptosis. Collectively, these data argue that the predisposition of Fancc-/- hematopoietic progenitors to apoptosis is mediated in part through altered redox regulation and Ask1 hyperactivation.
Blood 01/2006; 106(13):4124-30. · 9.90 Impact Factor
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Xiaxin Li,
Michelle M Le Beau,
Samantha Ciccone,
Feng-Chun Yang,
Brian Freie,
Shi Chen,
Jin Yuan,
Ping Hong,
Attilio Orazi, Laura S Haneline,
D Wade Clapp
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ABSTRACT: Current strategies for genetic therapy using Moloney retroviruses require ex vivo manipulation of hematopoietic cells to facilitate stable integration of the transgene. While many studies have evaluated the impact of ex vivo culture on normal murine and human stem/progenitor cells, the cellular consequences of ex vivo manipulation of stem cells with intrinsic defects in genome stability are incompletely understood. Here we show that ex vivo culture of Fancc(-/-) bone marrow cells results in a time-dependent increase in apoptosis of primitive Fancc(-/-) progenitor cells in conditions that promote the proliferation of wild-type stem/progenitor cells. Further, recipients reconstituted with the surviving Fancc(-/-) cells have a high incidence of cytogenetic abnormalities and myeloid malignancies that are associated with an acquired resistance to tumor necrosis factor alpha (TNF-alpha). Collectively, these data indicate that the intrinsic defects in the genomic stability of Fancc(-/-) stem/progenitor cells provide a selective pressure for cells that are resistant to apoptosis and have a propensity for the evolution to clonal hematopoiesis and malignancy. These studies could have implications for the design of genetic therapies for treatment of Fanconi anemia and potentially other genetic diseases with intrinsic defects in genome stability.
Blood 06/2005; 105(9):3465-71. · 9.90 Impact Factor
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ABSTRACT: Fanconi anemia (FA) is characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of many FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. Previous studies in FA murine models and in a phase 1 clinical trial suggest that myelopreparation is required for significant engraftment of exogenous, genetically corrected stem cells. Since myeloid progenitors from Fancc-/- mice and human Fanconi anemia group C protein (FANCC) patients have increased apoptosis in response to interferon gamma (IFN-gamma) in vitro, we hypothesized that IFN-gamma may be useful as a nongenotoxic, myelopreparative conditioning agent. To test this hypothesis, IFN-gamma was administered as a continuous infusion to Fancc-/- and wild-type (WT) mice for 1 week. Primitive and mature myeloid lineages were preferentially reduced in IFN-gamma-treated Fancc-/- mice. Further, IFN-gamma conditioning of Fancc-/- recipients was sufficient as a myelopreparative regimen to allow consistent engraftment of isogenic WT repopulating stem cells. Collectively, these data demonstrate that Fancc-/- hematopoietic cell populations have increased hypersensitivity to IFN-gamma in vivo and that IFN-gamma conditioning may be useful as a nongenotoxic strategy for myelopreparation in this disorder.
Blood 09/2004; 104(4):1204-9. · 9.90 Impact Factor
