Inhibition of FLT1 ameliorates muscular dystrophy phenotype by increased vasculature in a mouse model of Duchenne muscular dystrophy

Autoři: Mayank Verma aff001;  Yuko Shimizu-Motohashi aff002;  Yoko Asakura aff002;  James P. Ennen aff002;  Jennifer Bosco aff005;  Zhiwei Zhou aff005;  Guo-Hua Fong aff006;  Serene Josiah aff005;  Dennis Keefe aff005;  Atsushi Asakura aff002
Působiště autorů: Medical Scientist Training Program, University of Minnesota Medical School, Minneapolis, MN, United States of America aff001;  Stem Cell Institute, University of Minnesota Medical School, Minneapolis, MN, United States of America aff002;  Paul & Sheila Wellstone Muscular Dystrophy Center, University of Minnesota Medical School, Minneapolis, MN, United States of America aff003;  Department of Neurology, University of Minnesota Medical School, Minneapolis, MN, United States of America aff004;  Shire Human Genetic Therapies, Inc., a member of the Takeda group of companies, Lexington, MA, United States of America aff005;  Center for Vascular Biology, University of Connecticut Health Center, University of Connecticut School of Medicine, Farmington, CT, United States of America aff006
Vyšlo v časopise: Inhibition of FLT1 ameliorates muscular dystrophy phenotype by increased vasculature in a mouse model of Duchenne muscular dystrophy. PLoS Genet 15(12): e32767. doi:10.1371/journal.pgen.1008468
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008468


Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disease in which the dystrophin coding for a membrane stabilizing protein is mutated. Recently, the vasculature has also shown to be perturbed in DMD and DMD model mdx mice. Recent DMD transcriptomics revealed the defects were correlated to a vascular endothelial growth factor (VEGF) signaling pathway. To reveal the relationship between DMD and VEGF signaling, mdx mice were crossed with constitutive (CAGCreERTM:Flt1LoxP/LoxP) and endothelial cell-specific conditional gene knockout mice (Cdh5CreERT2:Flt1LoxP/LoxP) for Flt1 (VEGFR1) which is a decoy receptor for VEGF. Here, we showed that while constitutive deletion of Flt1 is detrimental to the skeletal muscle function, endothelial cell-specific Flt1 deletion resulted in increased vascular density, increased satellite cell number and improvement in the DMD-associated phenotype in the mdx mice. These decreases in pathology, including improved muscle histology and function, were recapitulated in mdx mice given anti-FLT1 peptides or monoclonal antibodies, which blocked VEGF-FLT1 binding. The histological and functional improvement of dystrophic muscle by FLT1 blockade provides a novel pharmacological strategy for the potential treatment of DMD.

Klíčová slova:

Body weight – Enzyme-linked immunoassays – Mouse models – Muscle fibers – Muscle functions – Skeletal muscles – Duchenne muscular dystrophy – Skeletal muscle fibers


1. Im W. B., Phelps S. F., Copen E. H., Adams E. G., Slightom J. L., Chamberlain J. S. Differential expression of dystrophin isoforms in strains of mdx mice with different mutations. Hum Mol Genet. 1996;5(8):1149–53. doi: 10.1093/hmg/5.8.1149 8842734.

2. Miyatake M, Miike T, Zhao J, Yoshioka K, Uchino M, Usuku G. Possible systemic smooth muscle layer dysfunction due to a deficiency of dystrophin in Duchenne muscular dystrophy. J Neurol Sci. 1989;93(1):11–7. doi: 10.1016/0022-510x(89)90157-3 2681539

3. Loufrani L, Matrougui K, Gorny D, Duriez M, Blanc I, Levy BI, et al. Flow (shear stress)-induced endothelium-dependent dilation is altered in mice lacking the gene encoding for dystrophin. Circulation. 2001;103:864–70. doi: 10.1161/01.cir.103.6.864 11171796

4. Ito K, Kimura S, Ozasa S, Matsukura M, Ikezawa M, Yoshioka K, et al. Smooth muscle-specific dystrophin expression improves aberrant vasoregulation in mdx mice. Hum Mol Genet. 2006;15(14):2266–75. doi: 10.1093/hmg/ddl151 16777842

5. Coral-Vazquez R, Cohn RD, Moore SA, Hill JA, Weiss RM, Davisson RL, et al. Disruption of the sarcoglycan-sarcospan complex in vascular smooth muscle: a novel mechanism for cardiomyopathy and muscular dystrophy. Cell. 1999;98(4):465–74. doi: 10.1016/s0092-8674(00)81975-3 10481911

6. Lai Y, Thomas GD, Yue Y, Yang HT, Li D, Long C, et al. Dystrophins carrying spectrin-like repeats 16 and 17 anchor nNOS to the sarcolemma and enhance exercise performance in a mouse model of muscular. J Clin Invest. 2009;119(3):624–35. doi: 10.1172/JCI36612 19229108

7. Asai A, Sahani N, Kaneki M, Ouchi Y, Martyn JA, Yasuhara SE. Primary role of functional ischemia, quantitative evidence for the two-hit mechanism, and phosphodiesterase-5 inhibitor therapy in mouse muscular dystrophy. PloS one. 2007;2(8):e806. Epub 2007/08/30. doi: 10.1371/journal.pone.0000806 17726536; PubMed Central PMCID: PMC1950086.

8. Adamo CM, Dai DF, Percival JM, Minami E, Willis MS, Patrucco E, et al. Sildenafil reverses cardiac dysfunction in the mdx mouse model of Duchenne muscular dystrophy. Proc Natl Acad Sci U S A. 2010;107:19079–83. doi: 10.1073/pnas.1013077107 20956307

9. Martin EA, Barresi R, Byrne BJ, Tsimerinov EI, Scott BL, Walker AE, et al. Tadalafil Alleviates Muscle Ischemia in Patients with Becker Muscular Dystrophy. Sci Transl Med. 2012;4(162):162ra55. doi: 10.1126/scitranslmed.3004327 23197572.

10. Nelson MD, Rader F, Tang X, Tavyev J, Nelson SF, Miceli MC, et al. PDE5 inhibition alleviates functional muscle ischemia in boys with Duchenne muscular dystrophy. Neurology. 2014:1–7. doi: 10.1212/WNL.0000000000000498 24808022.

11. Matsakas A, Yadav V, Lorca S, Narkar V. Muscle ERRγ mitigates Duchenne muscular dystrophy via metabolic and angiogenic reprogramming. FASEB J. 2013;27(10):4004–16. doi: 10.1096/fj.13-228296 23781095.

12. Latroche C, Matot B, Martins-Bach A, Briand D, Chazaud B, Wary C, et al. Structural and Functional Alterations of Skeletal Muscle Microvasculature in Dystrophin-Deficient mdx Mice. Am J Pathol. 2015:1–13. doi: 10.1016/j.ajpath.2015.05.009 26193666

13. Shibuya M. and dual function of vascular endothelial growth factor receptor-1 (Flt-1). Int J Biochem Cell Biol. 2001;33(4):409–20. doi: 10.1016/s1357-2725(01)00026-7 11312109.

14. Kendall RL, Thomas KA. Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor. Proc Natl Acad Sci U S A. 1993;90(22):10705–9. doi: 10.1073/pnas.90.22.10705 8248162; PubMed Central PMCID: PMC47846.

15. Verma M, Asakura Y, Hirai H, Watanabe S, Tastad C, Fong GH, et al. Flt-1 haploinsufficiency ameliorates muscular dystrophy phenotype by developmentally increased vasculature in mdx mice. Hum Mol Genet. 2010;19(21):4145–59. doi: 10.1093/hmg/ddq334 20705734.

16. Sawano a, Iwai S, Sakurai Y, Ito M, Shitara K, Nakahata T, et al. Flt-1, vascular endothelial growth factor receptor 1, is a novel cell surface marker for the lineage of monocyte-macrophages in humans. Blood. 2001;97:785–91. doi: 10.1182/blood.v97.3.785 11157498

17. Poesen K, Lambrechts D, Van Damme P, Dhondt J, Bender F, Frank N, et al. Novel role for vascular endothelial growth factor (VEGF) receptor-1 and its ligand VEGF-B in motor neuron degeneration. J Neurosci. 2008;28(42):10451–9. doi: 10.1523/JNEUROSCI.1092-08.2008 18923022; PubMed Central PMCID: PMC6671326.

18. Fong GH, Zhang L, Bryce DM, Peng J. Increased hemangioblast commitment, not vascular disorganization, is the primary defect in flt-1 knock-out mice. Development. 1999;126(13):3015–25. 10357944

19. Hayashi S, McMahon AP. Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev Biol. 2002;244:305–18. doi: 10.1006/dbio.2002.0597 11944939.

20. Ho VC, Duan LJ, Cronin C, Liang BT, Fong GH. Elevated vascular endothelial growth factor receptor-2 abundance contributes to increased angiogenesis in vascular endothelial growth factor receptor-1-deficient mice. Circulation. 2012;126(6):741–52. doi: 10.1161/CIRCULATIONAHA.112.091603 22753193.

21. Seki T, Hosaka K, Fischer C, Lim S, Andersson P, Abe M, et al. Ablation of endothelial VEGFR1 improves metabolic dysfunction by inducing adipose tissue browning. J Exp Med. 2018;215(2):611–26. doi: 10.1084/jem.20171012 29305395; PubMed Central PMCID: PMC5789413.

22. Waters RE, Rotevatn S, Li P, Annex BH, Yan Z. Voluntary running induces fiber type-specific angiogenesis in mouse skeletal muscle. Am J Physiol Cell Physiol. 2004;287(5):C1342–8. doi: 10.1152/ajpcell.00247.2004 15253894

23. Webster C, Silberstein L, Hays AP, Blau HM. Fast muscle fibers are preferentially affected in Duchenne muscular dystrophy. Cell. 1988;52(4):503–13. Epub 1988/02/26. doi: 10.1016/0092-8674(88)90463-1 3342447.

24. Rafael JA, Townsend ER, Squire SE, Potter AC, Chamberlain JS, Davies KE. Dystrophin and utrophin influence fiber type composition and post-synaptic membrane structure. Hum Mol Genet. 2000;9(9):1357–67. doi: 10.1093/hmg/9.9.1357 10814717.

25. Call JA, Warren GL, Verma M, Lowe DA. Acute failure of action potential conduction in mdx muscle reveals new mechanism of contraction-induced force loss. J Physiol. 2013;591(15):3765–76. doi: 10.1113/jphysiol.2013.254656 23753524.

26. Robciuc MR, Kivela R, Williams IM, de Boer JF, van Dijk TH, Elamaa H, et al. VEGFB/VEGFR1-Induced Expansion of Adipose Vasculature Counteracts Obesity and Related Metabolic Complications. Cell Metab. 2016;23(4):712–24. doi: 10.1016/j.cmet.2016.03.004 27076080; PubMed Central PMCID: PMC5898626.

27. Okabe K, Kobayashi S, Yamada T, Kurihara T, Tai-nagara I. Neurons Limit Angiogenesis by Titrating VEGF in Retina. Cell. 2014;159:584–96. doi: 10.1016/j.cell.2014.09.025 25417109

28. Goel AJ, Rieder MK, Arnold HH, Radice GL, Krauss RS. Niche Cadherins Control the Quiescence-to-Activation Transition in Muscle Stem Cells. Cell Rep. 2017;21(8):2236–50. doi: 10.1016/j.celrep.2017.10.102 29166613; PubMed Central PMCID: PMC5702939.

29. Kong J-S, Yoo S-A, Kang J-H, Ko W, Jeon S, Chae C-B, et al. Suppression of neovascularization and experimental arthritis by D-form of anti-flt-1 peptide conjugated with mini-PEG(™). Angiogenesis. 2011;14:431–42. doi: 10.1007/s10456-011-9226-0 21751011.

30. Liston DR, Davis M. Clinically Relevant Concentrations of Anticancer Drugs: A Guide for Nonclinical Studies. Clin Cancer Res. 2017;23(14):3489–98. doi: 10.1158/1078-0432.CCR-16-3083 28364015; PubMed Central PMCID: PMC5511563.

31. Christinger HW, Fuh G, de Vos AM, Wiesmann C. The crystal structure of placental growth factor in complex with domain 2 of vascular endothelial growth factor receptor-1. J Biol Chem. 2004;279(11):10382–8. doi: 10.1074/jbc.M313237200 14684734.

32. Springer ML, Ozawa CR, Banfi A, Kraft PE, Ip TK, Brazelton TR, et al. Localized arteriole formation directly adjacent to the site of VEGF-induced angiogenesis in muscle. Mol Ther. 2003;7(4):441–9. doi: 10.1016/s1525-0016(03)00010-8 12727106

33. Frank RT, Aboody KS, Najbauer J. Strategies for enhancing antibody delivery to the brain. Biochim Biophys Acta. 2011;1816(2):191–8. doi: 10.1016/j.bbcan.2011.07.002 21767610.

34. Lalatsa A, Schatzlein AG, Uchegbu IF. Strategies to deliver peptide drugs to the brain. Mol Pharm. 2014;11(4):1081–93. doi: 10.1021/mp400680d 24601686.

35. Miquerol L, Langille BL, Nagy A. Embryonic development is disrupted by modest increases in vascular endothelial growth factor gene expression. Development. 2000;127(18):3941–6. 10952892.

36. Gabhann FM, Ji JW, Popel AS. Computational model of vascular endothelial growth factor spatial distribution in muscle and pro-angiogenic cell therapy. PLoS Comput Biol. 2006;2:1107–20. doi: 10.1371/journal.pcbi.0020127 17002494.

37. Logsdon EA, Finley SD, Popel AS, F. MF. A systems biology view of blood vessel growth and remodelling. J Cell Mol Med. 2014;18:1491–508. doi: 10.1111/jcmm.12164 24237862.

38. Verma M, Asakura Y, Murakonda BSR, Pengo T, Latroche C, Chazaud B, et al. Muscle Satellite Cell Cross-Talk with a Vascular Niche Maintains Quiescence via VEGF and Notch Signaling. Cell Stem Cell. 2018;23(4):530–43 e9. doi: 10.1016/j.stem.2018.09.007 30290177; PubMed Central PMCID: PMC6178221.

39. Koch S, Tugues S, Li X, Gualandi L, Claesson-Welsh L. Signal transduction by vascular endothelial growth factor receptors. Biochem J. 2011;437:169–83. doi: 10.1042/BJ20110301 21711246.

40. Dewerchin M, Carmeliet P. PlGF: a multitasking cytokine with disease-restricted activity. Cold Spring Harb Perspect Med. 2012;2(8). doi: 10.1101/cshperspect.a011056 22908198; PubMed Central PMCID: PMC3405829.

41. Olofsson B, Pajusola K, Kaipainen A, von Euler G, Joukov V, Saksela O, et al. Vascular endothelial growth factor B, a novel growth factor for endothelial cells. Proc Natl Acad Sci U S A. 1996;93(6):2576–81. doi: 10.1073/pnas.93.6.2576 8637916; PubMed Central PMCID: PMC39839.

42. Hagberg CE, Mehlem A, Falkevall A, Muhl L, Fam BC, Ortsater H, et al. Targeting VEGF-B as a novel treatment for insulin resistance and type 2 diabetes. Nature. 2012;490(7420):426–30. doi: 10.1038/nature11464 23023133.

43. Li X, Tjwa M, Van Hove I, Enholm B, Neven E, Paavonen K, et al. Reevaluation of the role of VEGF-B suggests a restricted role in the revascularization of the ischemic myocardium. Arterioscler Thromb Vasc Biol. 2008;28(9):1614–20. doi: 10.1161/ATVBAHA.107.158725 18511699; PubMed Central PMCID: PMC2753879.

44. Messina S, Mazzeo A, Bitto A, Aguennouz M, Migliorato A, De Pasquale MG, et al. VEGF overexpression via adeno-associated virus gene transfer promotes skeletal muscle regeneration and enhances muscle function in mdx mice. FASEB J. 2007;21(13):3737–46. doi: 10.1096/fj.07-8459com 17575261.

45. Deasy BM, Feduska JM, Payne TR, Li Y, Ambrosio F, Huard J. Effect of VEGF on the regenerative capacity of muscle stem cells in dystrophic skeletal muscle. Mol Ther. 2009;17(10):1788–98. doi: 10.1038/mt.2009.136 19603004; PubMed Central PMCID: PMC2835014.

46. Gianni-Barrera R, Trani M, Fontanellaz C, Heberer M, Djonov V, Hlushchuk R, et al. VEGF over-expression in skeletal muscle induces angiogenesis by intussusception rather than sprouting. Angiogenesis. 2013;16:123–36. doi: 10.1007/s10456-012-9304-y 22961440.

47. Song X, Zhang Y, Hou Z, Wu H, Lu S, Tang J, et al. Adeno-associated virus serotype 9 mediated vascular endothelial growth factor gene overexpression in mdx mice. Exp Ther Med. 2018;15(2):1825–30. doi: 10.3892/etm.2017.5610 29434771; PubMed Central PMCID: PMC5776553.

48. Latroche C, Weiss-Gayet M, Muller L, Gitiaux C, Leblanc P, Liot S, et al. Coupling between Myogenesis and Angiogenesis during Skeletal Muscle Regeneration Is Stimulated by Restorative Macrophages. Stem Cell Reports. 2017;9(6):2018–33. doi: 10.1016/j.stemcr.2017.10.027 29198825; PubMed Central PMCID: PMC5785732.

49. Christov C, Chretien F, Abou-Khalil R, Bassez G, Vallet G, Authier FJ, et al. Muscle satellite cells and endothelial cells: close neighbors and privileged partners. Mol Biol Cell. 2007;18(4):1397–409. doi: 10.1091/mbc.E06-08-0693 17287398

50. Saitoh M, Ishida J, Ebner N, Anker SD, von Haehling S. Myostatin inhibitors as pharmacological treatment for muscle wasting and muscular dystrophy. JCSM Clin Reports 2017;2(1):e00037.

51. Wallace B, Peisl A, Seedorf G, Nowlin T, Kim C, Bosco J, et al. Anti-sFlt-1 Therapy Preserves Lung Alveolar and Vascular Growth in Antenatal Models of Bronchopulmonary Dysplasia. Am J Respir Crit Care Med. 2018;197(6):776–87. doi: 10.1164/rccm.201707-1371OC 29268623; PubMed Central PMCID: PMC5855071.

52. Danko I, Chapman V, Wolff JA. The frequency of revertants in mdx mouse genetic models for Duchenne muscular dystrophy. Pediatr Res. 1992;32(1):128–31. doi: 10.1203/00006450-199207000-00025 1635838

53. Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L. A global double-fluorescent Cre reporter mouse. Genesis. 2007;45(9):593–605. doi: 10.1002/dvg.20335 17868096.

54. Bae DG, Kim TD, Li G, Yoon WH, Chae CB. Anti-flt1 peptide, a vascular endothelial growth factor receptor 1-specific hexapeptide, inhibits tumor growth and metastasis. Clin Cancer Res. 2005;11:2651–61. doi: 10.1158/1078-0432.CCR-04-1564 15814646

55. Aartsma-Rus A, van Putten M. Assessing functional performance in the mdx mouse model. J Vis Exp. 2014;(85). doi: 10.3791/51303 24747372; PubMed Central PMCID: PMC4158772.

56. Fukada S, Morikawa D, Yamamoto Y, Yoshida T, Sumie N, Yamaguchi M, et al. Genetic background affects properties of satellite cells and mdx phenotypes. Am J Pathol. 2010;176:2414–24. doi: 10.2353/ajpath.2010.090887 20304955.

57. Jin Q, Qiao C, Li J, Xiao B, Li J, Xiao X. A GDF11/myostatin inhibitor, GDF11 propeptide-Fc, increases skeletal muscle mass and improves muscle strength in dystrophic mdx mice. Skelet Muscle. 2019;9(1):16. doi: 10.1186/s13395-019-0197-y 31133057; PubMed Central PMCID: PMC6537384.

58. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–82. doi: 10.1038/nmeth.2019 22743772.

Genetika Reprodukční medicína

Článek vyšel v časopise

PLOS Genetics

2019 Číslo 12

Nejčtenější v tomto čísle
Zapomenuté heslo

Nemáte účet?  Registrujte se

Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.


Nemáte účet?  Registrujte se