Chronic activation of endothelial MAPK disrupts hematopoiesis via NFKB dependent inflammatory stress reversible by SCGF.
Animals
Antigens, CD
Bone Marrow
Cadherins
Endothelial Cells
/ metabolism
Female
Hematopoiesis
/ physiology
Hematopoietic Cell Growth Factors
/ metabolism
Hematopoietic Stem Cell Transplantation
Hematopoietic Stem Cells
Inflammation
Lectins, C-Type
/ metabolism
Male
Mice
Mitogen-Activated Protein Kinase Kinases
/ metabolism
NF-kappa B
/ metabolism
Signal Transduction
Transplantation, Autologous
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
03 02 2020
03 02 2020
Historique:
received:
10
04
2019
accepted:
13
01
2020
entrez:
5
2
2020
pubmed:
6
2
2020
medline:
24
4
2020
Statut:
epublish
Résumé
Inflammatory signals arising from the microenvironment have emerged as critical regulators of hematopoietic stem cell (HSC) function during diverse processes including embryonic development, infectious diseases, and myelosuppressive injuries caused by irradiation and chemotherapy. However, the contributions of cellular subsets within the microenvironment that elicit niche-driven inflammation remain poorly understood. Here, we identify endothelial cells as a crucial component in driving bone marrow (BM) inflammation and HSC dysfunction observed following myelosuppression. We demonstrate that sustained activation of endothelial MAPK causes NF-κB-dependent inflammatory stress response within the BM, leading to significant HSC dysfunction including loss of engraftment ability and a myeloid-biased output. These phenotypes are resolved upon inhibition of endothelial NF-κB signaling. We identify SCGF as a niche-derived factor that suppresses BM inflammation and enhances hematopoietic recovery following myelosuppression. Our findings demonstrate that chronic endothelial inflammation adversely impacts niche activity and HSC function which is reversible upon suppression of inflammation.
Identifiants
pubmed: 32015345
doi: 10.1038/s41467-020-14478-8
pii: 10.1038/s41467-020-14478-8
pmc: PMC6997369
doi:
Substances chimiques
Antigens, CD
0
Cadherins
0
Hematopoietic Cell Growth Factors
0
Lectins, C-Type
0
NF-kappa B
0
cadherin 5
0
Mitogen-Activated Protein Kinase Kinases
EC 2.7.12.2
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
666Subventions
Organisme : NIA NIH HHS
ID : R01 AG065436
Pays : United States
Organisme : NIAMS NIH HHS
ID : R01 AR075585
Pays : United States
Organisme : NCI NIH HHS
ID : R01 CA204308
Pays : United States
Organisme : NHLBI NIH HHS
ID : R01 HL133021
Pays : United States
Références
Wagers, A. J. The stem cell niche in regenerative medicine. Cell Stem Cell 10, 362–369 (2012).
pubmed: 22482502
doi: 10.1016/j.stem.2012.02.018
Lane, S. W., Williams, D. A. & Watt, F. M. Modulating the stem cell niche for tissue regeneration. Nat. Biotechnol. 32, 795–803 (2014).
pubmed: 25093887
pmcid: 4422171
doi: 10.1038/nbt.2978
Schepers, K., Campbell, T. B. & Passegue, E. Normal and leukemic stem cell niches: insights and therapeutic opportunities. Cell Stem Cell 16, 254–267 (2015).
pubmed: 25748932
pmcid: 4391962
doi: 10.1016/j.stem.2015.02.014
Baldridge, M. T., King, K. Y. & Goodell, M. A. Inflammatory signals regulate hematopoietic stem cells. Trends Immunol. 32, 57–65 (2011).
pubmed: 21233016
pmcid: 3042730
doi: 10.1016/j.it.2010.12.003
Zhao, J. L. & Baltimore, D. Regulation of stress-induced hematopoiesis. Curr. Opin. Hematol. 22, 286–292 (2015).
pubmed: 26049748
pmcid: 4573392
doi: 10.1097/MOH.0000000000000149
Boettcher, S. & Manz, M. G. Regulation of inflammation- and infection-driven hematopoiesis. Trends Immunol. 38, 345–357 (2017).
pubmed: 28216309
doi: 10.1016/j.it.2017.01.004
Espin-Palazon, R., Weijts, B., Mulero, V. & Traver, D. Proinflammatory signals as fuel for the fire of hematopoietic stem cell emergence. Trends Cell Biol. 28, 58–66 (2018).
pubmed: 28882414
doi: 10.1016/j.tcb.2017.08.003
Bowers, E. et al. Granulocyte-derived TNFalpha promotes vascular and hematopoietic regeneration in the bone marrow. Nat. Med. 24, 95–102 (2018).
pubmed: 29155425
doi: 10.1038/nm.4448
Kovtonyuk, L. V., Fritsch, K., Feng, X., Manz, M. G. & Takizawa, H. Inflamm-aging of hematopoiesis, hematopoietic stem cells, and the bone marrow microenvironment. Front. Immunol. 7, 502 (2016).
pubmed: 27895645
pmcid: 5107568
doi: 10.3389/fimmu.2016.00502
Pietras, E. M. et al. Chronic interleukin-1 exposure drives haematopoietic stem cells towards precocious myeloid differentiation at the expense of self-renewal. Nat. Cell Biol. 18, 607–618 (2016).
pubmed: 27111842
pmcid: 4884136
doi: 10.1038/ncb3346
Lussana, F. & Rambaldi, A. Inflammation and myeloproliferative neoplasms. J. Autoimmun. 85, 58–63 (2017).
pubmed: 28669446
doi: 10.1016/j.jaut.2017.06.010
Pietras, E. M. Inflammation: a key regulator of hematopoietic stem cell fate in health and disease. Blood 130, 1693–1698 (2017).
pubmed: 28874349
pmcid: 5639485
doi: 10.1182/blood-2017-06-780882
Hooper, A. T. et al. Engraftment and reconstitution of hematopoiesis is dependent on VEGFR2-mediated regeneration of sinusoidal endothelial cells. Cell Stem Cell 4, 263–274 (2009).
pubmed: 19265665
pmcid: 3228275
doi: 10.1016/j.stem.2009.01.006
Butler, J. M. et al. Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. Cell Stem Cell 6, 251–264 (2010).
pubmed: 20207228
pmcid: 2866527
doi: 10.1016/j.stem.2010.02.001
Kobayashi, H. et al. Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells. Nat. Cell Biol. 12, 1046–1056 (2010).
pubmed: 20972423
pmcid: 2972406
doi: 10.1038/ncb2108
Winkler, I. G. et al. Vascular niche E-selectin regulates hematopoietic stem cell dormancy, self renewal and chemoresistance. Nat. Med. 18, 1651–1657 (2012).
pubmed: 23086476
doi: 10.1038/nm.2969
Ding, L., Saunders, T. L., Enikolopov, G. & Morrison, S. J. Endothelial and perivascular cells maintain haematopoietic stem cells. Nature 481, 457–462 (2012).
pubmed: 22281595
pmcid: 3270376
doi: 10.1038/nature10783
Poulos, M. G. et al. Endothelial jagged-1 is necessary for homeostatic and regenerative hematopoiesis. Cell Rep. 4, 1022–1034 (2013).
pubmed: 24012753
pmcid: 3805263
doi: 10.1016/j.celrep.2013.07.048
Greenbaum, A. et al. CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance. Nature 495, 227–230 (2013).
pubmed: 23434756
pmcid: 3600148
doi: 10.1038/nature11926
Doan, P. L. et al. Epidermal growth factor regulates hematopoietic regeneration after radiation injury. Nat. Med. 19, 295–304 (2013).
pubmed: 23377280
pmcid: 3594347
doi: 10.1038/nm.3070
Poulos, M. G. et al. Endothelial-specific inhibition of NF-kappaB enhances functional haematopoiesis. Nat. Commun. 7, 13829 (2016).
pubmed: 28000664
pmcid: 5187502
doi: 10.1038/ncomms13829
Kusumbe, A. P. et al. Age-dependent modulation of vascular niches for haematopoietic stem cells. Nature 532, 380–384 (2016).
pubmed: 27074508
pmcid: 5035541
doi: 10.1038/nature17638
Morrison, S. J. & Scadden, D. T. The bone marrow niche for haematopoietic stem cells. Nature 505, 327–334 (2014).
pubmed: 24429631
pmcid: 4514480
doi: 10.1038/nature12984
Rafii, S., Butler, J. M. & Ding, B. S. Angiocrine functions of organ-specific endothelial cells. Nature 529, 316–325 (2016).
pubmed: 26791722
pmcid: 4878406
doi: 10.1038/nature17040
Pober, J. S. & Sessa, W. C. Evolving functions of endothelial cells in inflammation. Nat. Rev. Immunol. 7, 803–815 (2007).
pubmed: 17893694
doi: 10.1038/nri2171
Boettcher, S. et al. Endothelial cells translate pathogen signals into G-CSF-driven emergency granulopoiesis. Blood 124, 1393–1403 (2014).
pubmed: 24990886
pmcid: 4148762
doi: 10.1182/blood-2014-04-570762
Wang, L. et al. Notch-dependent repression of miR-155 in the bone marrow niche regulates hematopoiesis in an NF-kappaB-dependent manner. Cell Stem Cell 15, 51–65 (2014).
pubmed: 24996169
pmcid: 4398997
doi: 10.1016/j.stem.2014.04.021
Sanchez, A. et al. Map3k8 controls granulocyte colony-stimulating factor production and neutrophil precursor proliferation in lipopolysaccharide-induced emergency granulopoiesis. Sci. Rep. 7, 5010 (2017).
pubmed: 28694430
pmcid: 5503936
doi: 10.1038/s41598-017-04538-3
Roth Flach, R. J. et al. Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis. Nat. Commun. 6, 8995 (2015).
pubmed: 26688060
pmcid: 4703891
doi: 10.1038/ncomms9995
Vandoorne, K. et al. Imaging the vascular bone marrow niche during inflammatory stress. Circ. Res. 123, 415–427 (2018).
pubmed: 29980569
pmcid: 6202141
doi: 10.1161/CIRCRESAHA.118.313302
Baker, R. G., Hayden, M. S. & Ghosh, S. NF-kappaB, inflammation, and metabolic disease. Cell Metab. 13, 11–22 (2011).
pubmed: 21195345
pmcid: 3040418
doi: 10.1016/j.cmet.2010.12.008
Bottero, V., Withoff, S. & Verma, I. M. NF-kappaB and the regulation of hematopoiesis. Cell Death Differ. 13, 785–797 (2006).
pubmed: 16528384
doi: 10.1038/sj.cdd.4401888
Brown, K., Park, S., Kanno, T., Franzoso, G. & Siebenlist, U. Mutual regulation of the transcriptional activator NF-kappa B and its inhibitor, I kappa B-alpha. Proc. Natl Acad. Sci. USA 90, 2532–2536 (1993).
pubmed: 8460169
doi: 10.1073/pnas.90.6.2532
Wu, C. & Ghosh, S. Differential phosphorylation of the signal-responsive domain of I kappa B alpha and I kappa B beta by I kappa B kinases. J. Biol. Chem. 278, 31980–31987 (2003).
pubmed: 12791687
doi: 10.1074/jbc.M304278200
pmcid: 12791687
Yang, F., Tang, E., Guan, K. & Wang, C. Y. IKK beta plays an essential role in the phosphorylation of RelA/p65 on serine 536 induced by lipopolysaccharide. J. Immunol. 170, 5630–5635 (2003).
pubmed: 12759443
doi: 10.4049/jimmunol.170.11.5630
pmcid: 12759443
Wessel, A. W. & Hanson, E. P. A method for the quantitative analysis of stimulation-induced nuclear translocation of the p65 subunit of NF-kappaB from patient-derived dermal fibroblasts. Methods Mol. Biol. 1280, 413–426 (2015).
pubmed: 25736764
pmcid: 5597957
doi: 10.1007/978-1-4939-2422-6_25
Boehm, J. S. et al. Integrative genomic approaches identify IKBKE as a breast cancer oncogene. Cell 129, 1065–1079 (2007).
pubmed: 17574021
doi: 10.1016/j.cell.2007.03.052
pmcid: 17574021
Brown, K., Gerstberger, S., Carlson, L., Franzoso, G. & Siebenlist, U. Control of I kappa B-alpha proteolysis by site-specific, signal-induced phosphorylation. Science 267, 1485–1488 (1995).
pubmed: 7878466
doi: 10.1126/science.7878466
Kisseleva, T. et al. NF-kappaB regulation of endothelial cell function during LPS-induced toxemia and cancer. J. Clin. Invest. 116, 2955–2963 (2006).
pubmed: 17053836
pmcid: 1616192
doi: 10.1172/JCI27392
Wei, Q. & Frenette, P. S. Niches for hematopoietic stem cells and their progeny. Immunity 48, 632–648 (2018).
pubmed: 29669248
pmcid: 6103525
doi: 10.1016/j.immuni.2018.03.024
Crane, G. M., Jeffery, E. & Morrison, S. J. Adult haematopoietic stem cell niches. Nat. Rev. Immunol. 17, 573–590 (2017).
pubmed: 28604734
doi: 10.1038/nri.2017.53
Comazzetto, S. et al. Restricted hematopoietic progenitors and erythropoiesis require SCF from leptin receptor+ niche cells in the bone marrow. Cell Stem Cell 24, 477–486.e476 (2019).
pubmed: 30661958
pmcid: 6813769
doi: 10.1016/j.stem.2018.11.022
Inra, C. N. et al. A perisinusoidal niche for extramedullary haematopoiesis in the spleen. Nature 527, 466–471 (2015).
pubmed: 26570997
pmcid: 4838203
doi: 10.1038/nature15530
Sorensen, I., Adams, R. H. & Gossler, A. DLL1-mediated Notch activation regulates endothelial identity in mouse fetal arteries. Blood 113, 5680–5688 (2009).
pubmed: 19144989
doi: 10.1182/blood-2008-08-174508
Langen, U. H. et al. Cell-matrix signals specify bone endothelial cells during developmental osteogenesis. Nat. Cell Biol. 19, 189–201 (2017).
pubmed: 28218908
pmcid: 5580829
doi: 10.1038/ncb3476
Payne, S., De Val, S. & Neal, A. Endothelial-specific Cre mouse models. Arterioscler Thromb. Vasc. Biol. 38, 2550–2561 (2018).
pubmed: 30354251
pmcid: 6218004
doi: 10.1161/ATVBAHA.118.309669
Kilani, B. et al. Comparison of endothelial promoter efficiency and specificity in mice reveals a subset of Pdgfb-positive hematopoietic cells. J. Thromb. Haemost. 17, 827–840 (2019).
pubmed: 30801958
doi: 10.1111/jth.14417
Forde, A., Constien, R., Grone, H. J., Hammerling, G. & Arnold, B. Temporal Cre-mediated recombination exclusively in endothelial cells using Tie2 regulatory elements. Genesis 33, 191–197 (2002).
pubmed: 12203917
doi: 10.1002/gene.10117
Tang, Y., Harrington, A., Yang, X., Friesel, R. E. & Liaw, L. The contribution of the Tie2+ lineage to primitive and definitive hematopoietic cells. Genesis 48, 563–567 (2010).
pubmed: 20645309
pmcid: 2944906
doi: 10.1002/dvg.20654
Gareus, R. et al. Endothelial cell-specific NF-kappaB inhibition protects mice from atherosclerosis. Cell Metab. 8, 372–383 (2008).
pubmed: 19046569
doi: 10.1016/j.cmet.2008.08.016
Korhonen, H. et al. Anaphylactic shock depends on endothelial Gq/G11. J. Exp. Med. 206, 411–420 (2009).
pubmed: 19171764
pmcid: 2646572
doi: 10.1084/jem.20082150
Bartels, K., Grenz, A. & Eltzschig, H. K. Hypoxia and inflammation are two sides of the same coin. Proc. Natl Acad. Sci. USA 110, 18351–18352 (2013).
pubmed: 24187149
doi: 10.1073/pnas.1318345110
Karhausen, J., Haase, V. H. & Colgan, S. P. Inflammatory hypoxia: role of hypoxia-inducible factor. Cell Cycle 4, 256–258 (2005).
pubmed: 15655360
doi: 10.4161/cc.4.2.1407
Eltzschig, H. K. & Carmeliet, P. Hypoxia and inflammation. N. Engl. J. Med. 364, 656–665 (2011).
pubmed: 21323543
pmcid: 3930928
doi: 10.1056/NEJMra0910283
Mittal, M., Siddiqui, M. R., Tran, K., Reddy, S. P. & Malik, A. B. Reactive oxygen species in inflammation and tissue injury. Antioxid. Redox Signal. 20, 1126–1167 (2014).
pubmed: 23991888
pmcid: 3929010
doi: 10.1089/ars.2012.5149
Bigarella, C. L., Liang, R. & Ghaffari, S. Stem cells and the impact of ROS signaling. Development 141, 4206–4218 (2014).
pubmed: 25371358
pmcid: 4302918
doi: 10.1242/dev.107086
Takubo, K. et al. Regulation of the HIF-1alpha level is essential for hematopoietic stem cells. Cell Stem Cell 7, 391–402 (2010).
pubmed: 20804974
doi: 10.1016/j.stem.2010.06.020
Ludin, A. et al. Reactive oxygen species regulate hematopoietic stem cell self-renewal, migration and development, as well as their bone marrow microenvironment. Antioxid. Redox Signal. 21, 1605–1619 (2014).
pubmed: 24762207
pmcid: 4175025
doi: 10.1089/ars.2014.5941
Zhou, B. O., Yue, R., Murphy, M. M., Peyer, J. G. & Morrison, S. J. Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow. Cell Stem Cell 15, 154–168 (2014).
pubmed: 24953181
pmcid: 4127103
doi: 10.1016/j.stem.2014.06.008
Ding, L. & Morrison, S. J. Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches. Nature 495, 231–235 (2013).
pubmed: 23434755
pmcid: 3600153
doi: 10.1038/nature11885
Mossadegh-Keller, N. et al. M-CSF instructs myeloid lineage fate in single haematopoietic stem cells. Nature 497, 239–243 (2013).
pubmed: 23575636
pmcid: 3679883
doi: 10.1038/nature12026
Yue, R., Shen, B. & Morrison, S. J. Clec11a/osteolectin is an osteogenic growth factor that promotes the maintenance of the adult skeleton. Elife 5, https://doi.org/10.7554/eLife.18782 (2016).
Keller, C. C. et al. Suppression of a novel hematopoietic mediator in children with severe malarial anemia. Infect. Immun. 77, 3864–3871 (2009).
pubmed: 19528216
pmcid: 2737985
doi: 10.1128/IAI.00342-09
Ito, C. et al. Serum stem cell growth factor for monitoring hematopoietic recovery following stem cell transplantation. Bone Marrow Transplant. 32, 391–398 (2003).
pubmed: 12900775
doi: 10.1038/sj.bmt.1704152
Mazumder, B., Li, X. & Barik, S. Translation control: a multifaceted regulator of inflammatory response. J. Immunol. 184, 3311–3319 (2010).
pubmed: 20304832
pmcid: 2860598
doi: 10.4049/jimmunol.0903778
McDermott, B. T., Peffers, M. J., McDonagh, B. & Tew, S. R. Translational regulation contributes to the secretory response of chondrocytic cells following exposure to Interleukin-1beta. J. Biol. Chem. https://doi.org/10.1074/jbc.RA118.006865 (2019).
doi: 10.1074/jbc.RA118.006865
pubmed: 31300557
pmcid: 6721953
Li, X. M., Hu, Z., Jorgenson, M. L., Wingard, J. R. & Slayton, W. B. Bone marrow sinusoidal endothelial cells undergo nonapoptotic cell death and are replaced by proliferating sinusoidal cells in situ to maintain the vascular niche following lethal irradiation. Exp. Hematol. 36, 1143–1156 (2008).
doi: 10.1016/j.exphem.2008.04.013
Baselet, B., Sonveaux, P., Baatout, S. & Aerts, A. Pathological effects of ionizing radiation: endothelial activation and dysfunction. Cell Mol. Life Sci. 76, 699–728 (2019).
pubmed: 30377700
doi: 10.1007/s00018-018-2956-z
Zhang, H. et al. Sepsis induces hematopoietic stem cell exhaustion and myelosuppression through distinct contributions of TRIF and MYD88. Stem Cell Rep. 6, 940–956 (2016).
doi: 10.1016/j.stemcr.2016.05.002
Mirantes, C., Passegue, E. & Pietras, E. M. Pro-inflammatory cytokines: emerging players regulating HSC function in normal and diseased hematopoiesis. Exp. Cell Res. 329, 248–254 (2014).
pubmed: 25149680
pmcid: 4250307
doi: 10.1016/j.yexcr.2014.08.017
Geest, C. R. & Coffer, P. J. MAPK signaling pathways in the regulation of hematopoiesis. J. Leukoc. Biol. 86, 237–250 (2009).
pubmed: 19498045
doi: 10.1189/jlb.0209097
Xiao, L., Liu, Y. & Wang, N. New paradigms in inflammatory signaling in vascular endothelial cells. Am. J. Physiol. Heart Circ. Physiol. 306, H317–H325 (2014).
pubmed: 24285111
doi: 10.1152/ajpheart.00182.2013
Korpela, E. & Liu, S. K. Endothelial perturbations and therapeutic strategies in normal tissue radiation damage. Radiat. Oncol. 9, 266 (2014).
pubmed: 25518850
pmcid: 4279961
doi: 10.1186/s13014-014-0266-7
Halle, M. et al. Sustained inflammation due to nuclear factor-kappa B activation in irradiated human arteries. J. Am. Coll. Cardiol. 55, 1227–1236 (2010).
pubmed: 20298930
doi: 10.1016/j.jacc.2009.10.047
Munshi, A. & Ramesh, R. Mitogen-activated protein kinases and their role in radiation response. Genes Cancer 4, 401–408 (2013).
pubmed: 24349638
pmcid: 3863336
doi: 10.1177/1947601913485414
Wang, Y., Liu, L. & Zhou, D. Inhibition of p38 MAPK attenuates ionizing radiation-induced hematopoietic cell senescence and residual bone marrow injury. Radiat. Res. 176, 743–752 (2011).
pubmed: 22014293
pmcid: 3390189
doi: 10.1667/RR2727.1
Dong, F. et al. Cadmium induces vascular permeability via activation of the p38 MAPK pathway. Biochem. Biophys. Res. Commun. 450, 447–452 (2014).
pubmed: 24909688
doi: 10.1016/j.bbrc.2014.05.140
Li, L. et al. P38/MAPK contributes to endothelial barrier dysfunction via MAP4 phosphorylation-dependent microtubule disassembly in inflammation-induced acute lung injury. Sci. Rep. 5, 8895 (2015).
pubmed: 25746230
pmcid: 4352893
doi: 10.1038/srep08895
Brown, G. D., Willment, J. A. & Whitehead, L. C-type lectins in immunity and homeostasis. Nat. Rev. Immunol. 18, 374–389 (2018).
pubmed: 29581532
doi: 10.1038/s41577-018-0004-8
pmcid: 29581532
Srinivasan, L. et al. PI3 kinase signals BCR-dependent mature B cell survival. Cell 139, 573–586 (2009).
pubmed: 19879843
pmcid: 2787092
doi: 10.1016/j.cell.2009.08.041
Benedito, R. et al. The notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis. Cell 137, 1124–1135 (2009).
pubmed: 19524514
doi: 10.1016/j.cell.2009.03.025
DeFalco, J. et al. Virus-assisted mapping of neural inputs to a feeding center in the hypothalamus. Science 291, 2608–2613 (2001).
pubmed: 11283374
doi: 10.1126/science.1056602
Hu, Y. & Smyth, G. K. ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J. Immunol. Methods 347, 70–78 (2009).
pubmed: 19567251
doi: 10.1016/j.jim.2009.06.008
Gold, L. et al. Aptamer-based multiplexed proteomic technology for biomarker discovery. PLoS ONE 5, e15004 (2010).
pubmed: 21165148
pmcid: 3000457
doi: 10.1371/journal.pone.0015004