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1.
FASEB J ; 35(2): e21227, 2021 02.
Article in English | MEDLINE | ID: mdl-33247500

ABSTRACT

Mutations in any of the genes encoding the four subunits of succinate dehydrogenase (SDH), a mitochondrial membrane-bound enzyme complex that is involved in both the tricarboxylic acid cycle and the electron transport chain, can lead to a variety of disorders. Recognized conditions with such mutations include Leigh syndrome and hereditary tumors such as pheochromocytoma and paraganglioma (PPGL), renal cell carcinoma, and gastrointestinal stromal tumor. Tumors appear in SDH mutation carriers with dominant inheritance due to loss of heterozygosity in susceptible cells. Here, we describe a mouse model intended to reproduce hereditary PPGL through Cre-mediated loss of SDHC in cells that express tyrosine hydroxylase (TH), a compartment where PPGL is known to originate. We report that while there is modest expansion of TH+ glomus cells in the carotid body upon SDHC loss, PPGL is not observed in such mice, even in the presence of a conditional dominant negative p53 protein and chronic hypoxia. Instead, we report an unexpected phenotype of nondiabetic obesity beginning at about 20 weeks of age. We hypothesize that this obesity is caused by TH+ cell loss or altered phenotype in key compartments of the central nervous system responsible for regulating feeding behavior, coupled with metabolic changes due to loss of peripheral catecholamine production.


Subject(s)
Adrenal Gland Neoplasms/genetics , Disease Models, Animal , Neoplastic Syndromes, Hereditary/genetics , Obesity/genetics , Phenotype , Pheochromocytoma/genetics , Succinate Dehydrogenase/genetics , Adrenal Gland Neoplasms/pathology , Animals , Carcinogenesis/genetics , Carcinogenesis/pathology , Male , Mice , Mice, Inbred C57BL , Neoplastic Syndromes, Hereditary/pathology , Obesity/pathology , Pheochromocytoma/pathology , Succinate Dehydrogenase/deficiency
2.
Elife ; 92020 12 08.
Article in English | MEDLINE | ID: mdl-33289482

ABSTRACT

Metabolic dysfunction underlies several chronic diseases, many of which are exacerbated by obesity. Dietary interventions can reverse metabolic declines and slow aging, although compliance issues remain paramount. 17α-estradiol treatment improves metabolic parameters and slows aging in male mice. The mechanisms by which 17α-estradiol elicits these benefits remain unresolved. Herein, we show that 17α-estradiol elicits similar genomic binding and transcriptional activation through estrogen receptor α (ERα) to that of 17ß-estradiol. In addition, we show that the ablation of ERα completely attenuates the beneficial metabolic effects of 17α-E2 in male mice. Our findings suggest that 17α-E2 may act through the liver and hypothalamus to improve metabolic parameters in male mice. Lastly, we also determined that 17α-E2 improves metabolic parameters in male rats, thereby proving that the beneficial effects of 17α-E2 are not limited to mice. Collectively, these studies suggest ERα may be a drug target for mitigating chronic diseases in male mammals.


Subject(s)
Estradiol/physiology , Estrogen Receptor alpha/physiology , Longevity , Animals , Female , Gene Expression Regulation/physiology , Hypothalamus/metabolism , Hypothalamus/physiology , Insulin Resistance/physiology , Liver/metabolism , Liver/physiology , Longevity/physiology , Male , Mice , Mice, Knockout , Rats
3.
Exp Gerontol ; 142: 111113, 2020 12.
Article in English | MEDLINE | ID: mdl-33065227

ABSTRACT

Menopause is a natural physiological process in older women that is associated with reduced estrogen production and results in increased risk for obesity, diabetes, and osteoporosis. 17α-estradiol (17α-E2) treatment in males, but not females, reverses several metabolic conditions associated with advancing age, highlighting sexually dimorphic actions on age-related pathologies. In this study we sought to determine if 17α-E2 could prevent ovariectomy (OVX)-mediated detriments on adiposity and bone parameters in females. Eight-week-old female C57BL/6J mice were subjected to SHAM or OVX surgery and received dietary 17α-E2 during a six-week intervention period. We observed that 17α-E2 prevented OVX-induced increases in body weight and adiposity. Similarly, uterine weight and luminal cell thickness were decreased by OVX and prevented by 17α-E2 treatment. Interestingly, 17α-E2 prevented OVX-induced declines in tibial metaphysis cancellous bone. And similarly, 17α-E2 improved bone density parameters in both tibia and femur cancellous bone, primarily in OVX mice. In contrast, to the effects on cancellous bone, cortical bone parameters were largely unaffected by OVX or 17α-E2. In the non-weight bearing lumbar vertebrae, OVX reduced trabecular thickness but not spacing, while 17α-E2 increased trabecular thickness and reduced spacing. Despite this, 17α-E2 did improve bone volume/tissue volume in lumbar vertebrae. Overall, we found that 17α-E2 prevented OVX-induced increases in adiposity and changes in bone mass and architecture, with minimal effects in SHAM-operated mice. We also observed that 17α-E2 rescued uterine tissue mass and lining morphology to control levels without inducing hypertrophy, suggesting that 17α-E2 could be considered as an adjunct to traditional hormone replacement therapies.


Subject(s)
Bone Density , Estradiol , Aged , Animals , Estradiol/pharmacology , Estrogens , Female , Humans , Mice , Mice, Inbred C57BL , Obesity , Organ Size , Ovariectomy , Rats , Rats, Sprague-Dawley
4.
Acta Physiol (Oxf) ; 228(3): e13394, 2020 03.
Article in English | MEDLINE | ID: mdl-31560161

ABSTRACT

AIM: Tieg1 is involved in multiple signalling pathways, human diseases, and is highly expressed in muscle where its functions are poorly understood. METHODS: We have utilized Tieg1 knockout (KO) mice to identify novel and important roles for this transcription factor in regulating muscle ultrastructure, metabolism and mitochondrial functions in the soleus and extensor digitorum longus (EDL) muscles. RNA sequencing, immunoblotting, transmission electron microscopy, MRI, NMR, histochemical and mitochondrial function assays were performed. RESULTS: Loss of Tieg1 expression resulted in altered sarcomere organization and a significant decrease in mitochondrial number. Histochemical analyses demonstrated an absence of succinate dehydrogenase staining and a decrease in cytochrome c oxidase (COX) enzyme activity in KO soleus with similar, but diminished, effects in the EDL. Decreased complex I, COX and citrate synthase (CS) activities were detected in the soleus muscle of KO mice indicating altered mitochondrial function. Complex I activity was also diminished in KO EDL. Significant decreases in CS and respiratory chain complex activities were identified in KO soleus. 1 H-NMR spectra revealed no significant metabolic difference between wild-type and KO muscles. However, 31 P spectra revealed a significant decrease in phosphocreatine and ATPγ. Altered expression of 279 genes, many of which play roles in mitochondrial and muscle function, were identified in KO soleus muscle. Ultimately, all of these changes resulted in an exercise intolerance phenotype in Tieg1 KO mice. CONCLUSION: Our findings have implicated novel roles for Tieg1 in muscle including regulation of gene expression, metabolic activity and organization of tissue ultrastructure. This muscle phenotype resembles diseases associated with exercise intolerance and myopathies of unknown consequence.


Subject(s)
DNA-Binding Proteins/metabolism , Mitochondria/metabolism , Muscle, Skeletal/metabolism , Muscles/metabolism , Transcription Factors/metabolism , Animals , DNA-Binding Proteins/genetics , Disease Models, Animal , Electron Transport Complex IV/metabolism , Female , Metabolome , Mice , Mice, Knockout , Oxidative Stress/physiology , Physical Conditioning, Animal/physiology , Succinate Dehydrogenase/metabolism , Transcription Factors/genetics
5.
Front Cell Dev Biol ; 7: 214, 2019.
Article in English | MEDLINE | ID: mdl-31632967

ABSTRACT

NKAP is a multi-functional nuclear protein that has been shown to be essential for hematopoiesis. Deletion of NKAP in hematopoietic stem cells (HSCs) was previously found to result in rapid lethality and hematopoietic failure. NKAP deficient cells also exhibited diminished proliferation and increased expression of the cyclin dependent kinase inhibitors (CDKIs) p19 Ink4d and p21 Cip1. To determine how dysregulation of CDKI expression contributes to the effects of NKAP deficiency, NKAP was deleted in mice also deficient in p19 Ink4d or p21 Cip1 using poly-IC treatment to induce Mx1-cre. Hematopoietic failure and lethality were not prevented by deficiency in either CDKI when NKAP was deleted. Inducible deletion of NKAP in cultured hematopoietic progenitors ex vivo resulted in a senescent phenotype and altered expression of numerous cell cycle regulators including the CDKI p16 INK4a. Interestingly, while combined deficiency in p16 INK4a and p21 Cip1 did not reverse the effect of NKAP deficiency on hematopoiesis in vivo, it did shift the consequence of NKAP deficiency from senescence to apoptosis in ex vivo cultures. These results suggest that NKAP may limit cellular stress that can trigger cell cycle withdrawal or cell death, a role critical for the maintenance of a viable pool of hematopoietic progenitors.

6.
Gastroenterology ; 153(2): 521-535.e20, 2017 08.
Article in English | MEDLINE | ID: mdl-28438610

ABSTRACT

BACKGROUND & AIMS: Depletion of interstitial cells of Cajal (ICCs) is common in diabetic gastroparesis. However, in approximately 20% of patients with diabetes, gastric emptying (GE) is accelerated. GE also occurs faster in obese individuals, and is associated with increased blood levels of glucose in patients with type 2 diabetes. To understand the fate of ICCs in hyperinsulinemic, hyperglycemic states characterized by rapid GE, we studied mice with mutation of the leptin receptor (Leprdb/db), which in our colony had accelerated GE. We also investigated hyperglycemia-induced signaling in the ICC lineage and ICC dependence on glucose oxidative metabolism in mice with disruption of the succinate dehydrogenase complex, subunit C gene (Sdhc). METHODS: Mice were given breath tests to analyze GE of solids. ICCs were studied by flow cytometry, intracellular electrophysiology, isometric contractility measurement, reverse-transcription polymerase chain reaction, immunoblot, immunohistochemistry, enzyme-linked immunosorbent assays, and metabolite assays; cells and tissues were manipulated pharmacologically and by RNA interference. Viable cell counts, proliferation, and apoptosis were determined by methyltetrazolium, Ki-67, proliferating cell nuclear antigen, bromodeoxyuridine, and caspase-Glo 3/7 assays. Sdhc was disrupted in 2 different strains of mice via cre recombinase. RESULTS: In obese, hyperglycemic, hyperinsulinemic female Leprdb/db mice, GE was accelerated and gastric ICC and phasic cholinergic responses were increased. Female KitK641E/+ mice, which have genetically induced hyperplasia of ICCs, also had accelerated GE. In isolated cells of the ICC lineage and gastric organotypic cultures, hyperglycemia stimulated proliferation by mitogen-activated protein kinase 1 (MAPK1)- and MAPK3-dependent stabilization of ets variant 1-a master transcription factor for ICCs-and consequent up-regulation of v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) receptor tyrosine kinase. Opposite changes occurred in mice with disruption of Sdhc. CONCLUSIONS: Hyperglycemia increases ICCs via oxidative metabolism-dependent, MAPK1- and MAPK3-mediated stabilization of ets variant 1 and increased expression of KIT, causing rapid GE. Increases in ICCs might contribute to the acceleration in GE observed in some patients with diabetes.


Subject(s)
DNA-Binding Proteins/physiology , Gastric Emptying/physiology , Hyperglycemia/physiopathology , Interstitial Cells of Cajal/cytology , MAP Kinase Signaling System/physiology , Proto-Oncogene Proteins c-kit/physiology , Transcription Factors/physiology , Animals , Female , Humans , Interstitial Cells of Cajal/physiology , Mice , Mitogen-Activated Protein Kinase 1/physiology , Mitogen-Activated Protein Kinase 3/physiology , Receptors, Leptin/genetics , Up-Regulation
7.
Nucleic Acids Res ; 45(9): 5170-5182, 2017 May 19.
Article in English | MEDLINE | ID: mdl-28201653

ABSTRACT

We have previously demonstrated that TGFß Inducible Early Gene-1 (TIEG1), also known as KLF10, plays important roles in mediating skeletal development and homeostasis in mice. TIEG1 has also been identified in clinical studies as one of a handful of genes whose altered expression levels or allelic variations are associated with decreased bone mass and osteoporosis in humans. Here, we provide evidence for the first time that TIEG1 is involved in regulating the canonical Wnt signaling pathway in bone through multiple mechanisms of action. Decreased Wnt signaling in the absence of TIEG1 expression is shown to be in part due to impaired ß-catenin nuclear localization resulting from alterations in the activity of AKT and GSK-3ß. We also provide evidence that TIEG1 interacts with, and serves as a transcriptional co-activator for, Lef1 and ß-catenin. Changes in Wnt signaling in the setting of altered TIEG1 expression and/or activity may in part explain the observed osteopenic phenotype of TIEG1 KO mice as well as the known links between TIEG1 expression levels/allelic variations and patients with osteoporosis.


Subject(s)
Bone and Bones/metabolism , DNA-Binding Proteins/metabolism , Transcription Factors/metabolism , Wnt Signaling Pathway , beta Catenin/metabolism , Animals , Bone and Bones/drug effects , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Female , Gene Expression Regulation/drug effects , Ligands , Lithium Chloride/pharmacology , Lymphoid Enhancer-Binding Factor 1/metabolism , Mice, Inbred C57BL , Mice, Knockout , Models, Biological , Osteoblasts/drug effects , Osteoblasts/metabolism , Protein Binding/drug effects , Protein Transport/drug effects , Skull/cytology , Subcellular Fractions/drug effects , Subcellular Fractions/metabolism , Wnt Signaling Pathway/drug effects , Wnt Signaling Pathway/genetics
8.
PLoS One ; 10(5): e0127471, 2015.
Article in English | MEDLINE | ID: mdl-25985299

ABSTRACT

Familial paraganglioma (PGL) is a rare neuroendocrine cancer associated with defects in the genes encoding the subunits of succinate dehydrogenase (SDH), a tricarboxylic acid (TCA) cycle enzyme. For unknown reasons, a higher prevalence of PGL has been reported for humans living at higher altitude, with increased disease aggressiveness and morbidity. In this study, we evaluate the effects of oxygen on epigenetic changes due to succinate accumulation in three SDH loss cell culture models. We test the hypothesis that the mechanism of α-ketoglutarate (α-KG)-dependent dioxygenase enzymes explains the inhibitory synergy of hypoxia and succinate accumulation. We confirm that SDH loss leads to profound succinate accumulation. We further show that hypoxia and succinate accumulation synergistically inhibit α-KG-dependent dioxygenases leading to increased stabilization of transcription factor HIF1α, HIF2α, and hypermethylation of histones and DNA. Increasing oxygen suppresses succinate inhibition of α-KG-dependent dioxygenases. This result provides a possible explanation for the association between hypoxia and PGL, and suggests hyperoxia as a potential novel therapy.


Subject(s)
Epigenesis, Genetic , Models, Biological , Oxygen/metabolism , Paraganglioma/genetics , 5-Methylcytosine/analogs & derivatives , Animals , Case-Control Studies , Cytosine/analogs & derivatives , Cytosine/metabolism , DNA Methylation/drug effects , Dioxygenases/antagonists & inhibitors , Dioxygenases/metabolism , Epigenesis, Genetic/drug effects , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Knockdown Techniques , HEK293 Cells , Histones/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Ketoglutaric Acids/metabolism , Mice, Inbred C57BL , Succinate Dehydrogenase/metabolism , Succinic Acid/pharmacology
9.
Nucleic Acids Res ; 42(14): 8996-9004, 2014 Aug.
Article in English | MEDLINE | ID: mdl-25063301

ABSTRACT

The regulation of chromatin structure in eukaryotic cells involves abundant architectural factors such as high mobility group B (HMGB) proteins. It is not understood how these factors control the interplay between genome accessibility and compaction. In vivo, HMO1 binds the promoter and coding regions of most ribosomal RNA genes, facilitating transcription and possibly stabilizing chromatin in the absence of histones. To understand how HMO1 performs these functions, we combine single molecule stretching and atomic force microscopy (AFM). By stretching HMO1-bound DNA, we demonstrate a hierarchical organization of interactions, in which HMO1 initially compacts DNA on a timescale of seconds, followed by bridge formation and stabilization of DNA loops on a timescale of minutes. AFM experiments demonstrate DNA bridging between strands as well as looping by HMO1. Our results support a model in which HMO1 maintains the stability of nucleosome-free chromatin regions by forming complex and dynamic DNA structures mediated by protein-protein interactions.


Subject(s)
Chromatin/chemistry , DNA/chemistry , High Mobility Group Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , DNA/metabolism , DNA/ultrastructure , Nucleic Acid Conformation , Nucleosomes/chemistry
10.
PLoS One ; 8(5): e63491, 2013.
Article in English | MEDLINE | ID: mdl-23671681

ABSTRACT

ß-cell replacement may efficiently cure type 1 diabetic (T1D) patients whose insulin-secreting ß-cells have been selectively destroyed by autoantigen-reactive T cells. To generate insulin-secreting cells we used two cell sources: rat multipotent adult progenitor cells (rMAPC) and the highly similar rat extra-embryonic endoderm precursor (rXEN-P) cells isolated under rMAPC conditions from blastocysts (rHypoSC). rMAPC/rHypoSC were sequentially committed to definitive endoderm, pancreatic endoderm, and ß-cell like cells. On day 21, 20% of rMAPC/rHypoSC progeny expressed Pdx1 and C-peptide. rMAPCr/HypoSC progeny secreted C-peptide under the stimulus of insulin agonist carbachol, and was inhibited by the L-type voltage-dependent calcium channel blocker nifedipine. When rMAPC or rHypoSC differentiated d21 progeny were grafted under the kidney capsule of streptozotocin-induced diabetic nude mice, hyperglycemia reversed after 4 weeks in 6/10 rMAPC- and 5/10 rHypoSC-transplanted mice. Hyperglycemia recurred within 24 hours of graft removal and the histological analysis of the retrieved grafts revealed presence of Pdx1-, Nkx6.1- and C-peptide-positive cells. The ability of both rMAPC and HypoSC to differentiate to functional ß-cell like cells may serve to gain insight into signals that govern ß-cell differentiation and aid in developing culture systems to commit other (pluripotent) stem cells to clinically useful ß-cells for cell therapy of T1D.


Subject(s)
Blastocyst/cytology , Bone Marrow Cells/cytology , Germ Layers/transplantation , Hyperglycemia/surgery , Insulin-Secreting Cells/transplantation , Animals , Blastocyst/metabolism , Blotting, Western , Bone Marrow Cells/metabolism , C-Peptide/genetics , C-Peptide/metabolism , Cell Differentiation/genetics , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/surgery , Diabetes Mellitus, Type 1/complications , Diabetes Mellitus, Type 1/surgery , Embryonic Stem Cells/metabolism , Embryonic Stem Cells/transplantation , Endoderm/cytology , Endoderm/metabolism , Gene Expression , Germ Layers/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Hyperglycemia/complications , Insulin-Secreting Cells/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Multipotent Stem Cells/metabolism , Multipotent Stem Cells/transplantation , Rats , Rats, Inbred F344 , Reverse Transcriptase Polymerase Chain Reaction , Stem Cell Transplantation/methods , Time Factors , Trans-Activators/genetics , Trans-Activators/metabolism
11.
Nat Commun ; 4: 1582, 2013.
Article in English | MEDLINE | ID: mdl-23481390

ABSTRACT

Invariant natural killer T cells have a distinct developmental pathway from conventional αß T cells. Here we demonstrate that the transcriptional repressor NKAP is required for invariant natural killer T cell but not conventional T cell development. In CD4-cre NKAP conditional knockout mice, invariant natural killer T cell development is blocked at the double-positive stage. This cell-intrinsic block is not due to decreased survival or failure to rearrange the invariant Vα14-Jα18 T cell receptor-α chain, but is rescued by overexpression of a rec-Vα14-Jα18 transgene at the double-positive stage, thus defining a role for NKAP in selection into the invariant natural killer T cell lineage. Importantly, deletion of the NKAP-associated protein histone deacetylase 3 causes a similar block in the invariant natural killer T cell development, indicating that NKAP and histone deacetylase 3 functionally interact to control invariant natural killer T cell development.


Subject(s)
Natural Killer T-Cells/cytology , Repressor Proteins/metabolism , Animals , Cell Survival , Gene Deletion , Gene Rearrangement, alpha-Chain T-Cell Antigen Receptor , Histone Deacetylases/metabolism , Mice , Mice, Knockout , Natural Killer T-Cells/metabolism , Organ Specificity , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Receptors, Notch/metabolism , Recombination, Genetic/genetics , Repressor Proteins/deficiency , Thymocytes/cytology , Thymocytes/metabolism
12.
J Exp Med ; 208(6): 1291-304, 2011 Jun 06.
Article in English | MEDLINE | ID: mdl-21624937

ABSTRACT

Newly generated T cells are unable to respond to antigen/MHC. Rather, post-selection single-positive thymocytes must undergo T cell maturation to gain functional competency and enter the long-lived naive peripheral T cell pool. This process is poorly understood, as no gene specifically required for T cell maturation has been identified. Here, we demonstrate that loss of the transcriptional repressor NKAP results in a complete block in T cell maturation. In CD4-cre NKAP conditional knockout mice, thymic development including positive selection occurs normally, but there is a cell-intrinsic defect in the peripheral T cell pool. All peripheral naive CD4-cre NKAP conditional knockout T cells were found to be functionally immature recent thymic emigrants. This defect is not simply in cell survival, as the T cell maturation defect was not rescued by a Bcl-2 transgene. Thus, NKAP is required for T cell maturation and the acquisition of functional competency.


Subject(s)
Co-Repressor Proteins/metabolism , Nuclear Proteins/metabolism , Repressor Proteins/metabolism , T-Lymphocytes/cytology , Animals , CD4-Positive T-Lymphocytes/cytology , Cell Differentiation , Cell Separation , Flow Cytometry , Gene Deletion , Humans , Interleukin-7 Receptor alpha Subunit/metabolism , Mice , Mice, Knockout , Mice, Transgenic , Proto-Oncogene Proteins c-bcl-2/metabolism , Thymus Gland/cytology , Transgenes
13.
Blood ; 116(15): 2684-93, 2010 Oct 14.
Article in English | MEDLINE | ID: mdl-20610818

ABSTRACT

Steady-state hematopoiesis is sustained through differentiation balanced with proliferation and self-renewal of hematopoietic stem cells (HSCs). Disruption of this balance can lead to hematopoietic failure, as hematopoietic differentiation without self-renewal leads to loss of the HSC pool. We find that conditional knockout mice that delete the transcriptional repressor NKAP in HSCs and all hematopoietic lineages during embryonic development exhibit perinatal lethality and abrogation of hematopoiesis as demonstrated by multilineage defects in lymphocyte, granulocyte, erythrocyte and megakaryocyte development. Inducible deletion of NKAP in adult mice leads to lethality within 2 weeks, at which point hematopoiesis in the bone marrow has halted and HSCs have disappeared. This hematopoietic failure and lethality is cell intrinsic, as radiation chimeras reconstituted with inducible Mx1-cre NKAP conditional knockout bone marrow also succumb with a similar time course. Even in the context of a completely normal bone marrow environment using mixed radiation chimeras, NKAP deletion results in HSC failure. NKAP deletion leads to decreased proliferation and increased apoptosis of HSCs, which is likely due to increased expression of the cyclin-dependent kinase inhibitors p21Cip1/Waf1 and p19Ink4d. These data establish NKAP as one of a very small number of transcriptional regulators that is absolutely required for adult HSC maintenance and survival.


Subject(s)
Adult Stem Cells/cytology , Adult Stem Cells/metabolism , Co-Repressor Proteins/metabolism , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Animals , Animals, Newborn , Cell Proliferation , Cell Survival/genetics , Cell Survival/physiology , Co-Repressor Proteins/genetics , Cyclin-Dependent Kinase Inhibitor p21/genetics , Female , Hematopoiesis/genetics , Hematopoiesis/physiology , Male , Mice , Mice, Knockout , Mice, Transgenic , NF-kappa B/metabolism , Pregnancy , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Radiation Chimera
14.
Transplantation ; 85(10): 1489-95, 2008 May 27.
Article in English | MEDLINE | ID: mdl-18497691

ABSTRACT

BACKGROUND: Xenograft rejection can be provoked by both the innate and adaptive immune compartments and close reciprocal interactions exist between these two systems. We investigated the interdependent roles of T and B lymphocytes in vascularized (heart) and cellular (islet) xenograft rejection in a model with established xeno-nonreactivity of the innate immune system. METHODS: Specific innate xenotolerance was induced in nude rats bearing either a hamster heart or a hamster pancreatic islet graft by a tolerizing regimen consisting of donor antigen infusion, temporary natural killer cell depletion and a 4-week administration of leflunomide. One month after transplantation, syngeneic CD4 and CD8 T cells were adoptively transferred. RESULTS: Both vascular and cellular xenografts were rejected after CD4 T cell reconstitution, corresponding with production of high IgM and IgG xenoantibody titers. Deposition of xenoantibodies and complement was seen in the heart but not in the islet xenografts. After infusion of CD8 T cells, xenohearts underwent a delayed type of rejection without xenoantibody production and xenoislets were not rejected. In xenoislet recipients, CD8 dependent B cells were not tolerized, resulting in the production of IgG xenoantibodies belonging to Th2-dependent isotypes, known not to cause graft rejection, and deposited at the graft implantation site. CONCLUSIONS: We conclude that distinct mechanisms of immune activation underlie xenogeneic reactions against vascular and cellular grafts.


Subject(s)
Antibodies, Heterophile/immunology , CD8-Positive T-Lymphocytes/immunology , Heart Transplantation/immunology , Immune Tolerance/immunology , Islets of Langerhans Transplantation/immunology , T-Lymphocytes/immunology , Transplantation, Heterologous/immunology , Animals , Cricetinae , Graft Survival , Organ Specificity , Rats , Rats, Nude , Time Factors
15.
J Clin Invest ; 118(2): 505-14, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18172550

ABSTRACT

Despite progress in cardiovascular research, a cure for peripheral vascular disease has not been found. We compared the vascularization and tissue regeneration potential of murine and human undifferentiated multipotent adult progenitor cells (mMAPC-U and hMAPC-U), murine MAPC-derived vascular progenitors (mMAPC-VP), and unselected murine BM cells (mBMCs) in mice with moderate limb ischemia, reminiscent of intermittent claudication in human patients. mMAPC-U durably restored blood flow and muscle function and stimulated muscle regeneration, by direct and trophic contribution to vascular and skeletal muscle growth. This was in contrast to mBMCs and mMAPC-VP, which did not affect muscle regeneration and provided only limited and transient improvement. Moreover, mBMCs participated in a sustained inflammatory response in the lower limb, associated with progressive deterioration in muscle function. Importantly, mMAPC-U and hMAPC-U also remedied vascular and muscular deficiency in severe limb ischemia, representative of critical limb ischemia in humans. Thus, unlike BMCs or vascular-committed progenitors, undifferentiated multipotent adult progenitor cells offer the potential to durably repair ischemic damage in peripheral vascular disease patients.


Subject(s)
Extremities/blood supply , Ischemia/therapy , Multipotent Stem Cells/transplantation , Animals , Blood Vessels/cytology , Bone Marrow Transplantation , Cell Differentiation , Humans , Male , Mice , Mice, Inbred C57BL , Multipotent Stem Cells/cytology , Muscle Cells/cytology
16.
J Tissue Eng Regen Med ; 1(1): 51-9, 2007.
Article in English | MEDLINE | ID: mdl-18038392

ABSTRACT

Progressive contractile dysfunction of viable myocardium that surrounds a large infarct leads to heart failure following acute myocardial infarction (AMI). Experimental evidence indicates that cellular transplantation may improve the left ventricular (LV) contractile performance, even though the underlying mechanisms remain undefined. Here, we compared the effect of transplantation of murine multipotent adult progenitor cells (MAPCs), a population of adult bone marrow-derived cells that differentiate into cells of mesodermal, endodermal and ectodermal origin, with murine bone marrow cells (BMCs) or fibroblasts on post-infarct cardiac function by peri-infarct injection after coronary artery ligation in mice. We demonstrate that, in contrast to the other cell populations, transplantation of MAPCs significantly improved LV contractile function for at least 8 weeks post-transplantation and, although BMCs reduced infarct size, the decrease in scar size was substantially greater in MAPC-treated hearts. As neither MAPCs nor BMCs were present beyond 1 week, the beneficial effect was not due to differentiation and direct contribution of MAPCs to the vascular or cardiomyocyte compartment. Significantly more inflammatory cells were present in MAPC- than BMC-treated hearts at 1 week, which was accompanied by increased vascularity 8 weeks post-transplantation. We hypothesize that MAPCs indirectly contributed to these effects, by secreting inflammatory [monocyte chemoattractant protein-1 (MCP)-1], and vascular growth factors [vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF)-BB, and transforming growth factor (TGF)beta(1)), and others, resulting in increased angiogenensis and cardioprotection.


Subject(s)
Aging/physiology , Myocardial Infarction/physiopathology , Myocardial Infarction/surgery , Stem Cell Transplantation , Ventricular Function, Left/physiology , Animals , Cell Differentiation , Cell Proliferation , Cells, Cultured , Mice , Myocardial Infarction/pathology
17.
J Exp Med ; 204(1): 129-39, 2007 Jan 22.
Article in English | MEDLINE | ID: mdl-17227908

ABSTRACT

For decades, in vitro expansion of transplantable hematopoietic stem cells (HSCs) has been an elusive goal. Here, we demonstrate that multipotent adult progenitor cells (MAPCs), isolated from green fluorescent protein (GFP)-transgenic mice and expanded in vitro for >40-80 population doublings, are capable of multilineage hematopoietic engraftment of immunodeficient mice. Among MAPC-derived GFP+CD45.2+ cells in the bone marrow of engrafted mice, HSCs were present that could radioprotect and reconstitute multilineage hematopoiesis in secondary and tertiary recipients, as well as myeloid and lymphoid hematopoietic progenitor subsets and functional GFP+ MAPC-derived lymphocytes that were functional. Although hematopoietic contribution by MAPCs was comparable to control KTLS HSCs, approximately 10(3)-fold more MAPCs were required for efficient engraftment. Because GFP+ host-derived CD45.1+ cells were not observed, fusion is not likely to account for the generation of HSCs by MAPCs.


Subject(s)
Hematopoiesis , Hematopoietic Stem Cell Transplantation , Multipotent Stem Cells/transplantation , Animals , B-Lymphocytes/immunology , Graft Survival , Green Fluorescent Proteins/genetics , Hematopoiesis/immunology , Hematopoietic System/cytology , In Vitro Techniques , Lymphoid Tissue/cytology , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Multipotent Stem Cells/immunology , Organ Specificity , Recombinant Proteins/genetics , T-Lymphocytes/immunology
18.
Exp Hematol ; 34(11): 1596-601, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17046581

ABSTRACT

We describe methods for isolation of multipotent adult progenitor cells (MAPCs) from newborn to 6-week-old mice and rats. The maintenance of these cells, including their culture, media formulas, and quality control procedures, are also explained. Additionally, ways to identify MAPCs including their phenotype and morphology are discussed.


Subject(s)
Cell Separation/methods , Multipotent Stem Cells/cytology , Animals , Cell Culture Techniques/methods , Cell Differentiation/drug effects , Cell Differentiation/immunology , Cells, Cultured , Culture Media/pharmacology , Freezing , Immunohistochemistry , Mice , Multipotent Stem Cells/drug effects , Multipotent Stem Cells/immunology , Phenotype , Quality Control , Rats , Tissue Preservation
19.
Stem Cells ; 24(4): 1121-7, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16373692

ABSTRACT

Thymidine analogs, including bromodeoxyuridine, chlorodeoxyuridine, iododeoxyuridine, and tritiated thymidine, label dividing cells by incorporating into DNA during S phase of cell division and are widely employed to identify cells transplanted into the central nervous system. However, the potential for transfer of thymidine analogs from grafted cells to dividing host cells has not been thoroughly tested. We here demonstrate that graft-derived thymidine analogs can become incorporated into host neural precursors and glia. Large numbers of labeled neurons and glia were found 3-12 weeks after transplantation of thymidine analog-labeled live stem cells, suggesting differentiation of grafted cells. Remarkably, however, similar results were obtained after transplantation of dead cells or labeled fibroblasts. Our findings reveal for the first time that thymidine analog labeling may not be a reliable means of identifying transplanted cells, particularly in highly proliferative environments such as the developing, neurogenic, or injured brain.


Subject(s)
Central Nervous System/cytology , Central Nervous System/metabolism , Stem Cell Transplantation , Thymidine/analogs & derivatives , Animals , Animals, Newborn , Biological Transport, Active , Brain/cytology , Brain/growth & development , Brain/metabolism , Bromodeoxyuridine/pharmacokinetics , Cell Proliferation , Female , In Vitro Techniques , Mice , Mice, Transgenic , Neuroglia/metabolism , Neurons/metabolism , Pregnancy , Rats , Rats, Inbred SHR , Rats, Sprague-Dawley , Thymidine/pharmacokinetics
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