Skip to main content

Advertisement

SpringerLink
Log in

Isolation of human lymphatic malformation endothelial cells, their in vitro characterization and in vivo survival in a mouse xenograft model

  • Original Paper
  • Published:
Angiogenesis Aims and scope Submit manuscript

Abstract

Human lymphatic vascular malformations (LMs), also known as cystic hygromas or lymphangioma, consist of multiple lymphatic endothelial cell-lined lymph-containing cysts. No animal model of this disease exists. To develop a mouse xenograft model of human LM, CD34NegCD31Pos LM lymphatic endothelial cells (LM-LEC) were isolated from surgical specimens and compared to foreskin CD34NegCD31Pos lymphatic endothelial cells (LECs). Cells were implanted into a mouse tissue engineering model for 1, 2 and 4 weeks. In vitro LM-LECs showed increased proliferation and survival under starvation conditions (P < 0.0005 at 48 h, two-way ANOVA), increased migration (P < 0.001, two-way ANOVA) and formed fewer (P = 0.029, independent samples t test), shorter tubes (P = 0.029, independent samples t test) than foreskin LECs. In vivo LM-LECs implanted into a Matrigel™-containing mouse chamber model assembled to develop vessels with dilated cystic lumens lined with flat endothelium, morphology similar to that of clinical LMs. Human foreskin LECs failed to survive implantation. In LM-LEC implanted chambers the percent volume of podoplaninPos vessels was 1.18 ± 2.24 % at 1 week, 6.34 ± 2.68 % at 2 weeks and increasing to 7.67 ± 3.60 % at 4 weeks. In conclusion, the significantly increased proliferation, migration, resistance to apoptosis and decreased tubulogenesis of LM-LECs observed in vitro is likely to account for their survival and assembly into stable LM-like structures when implanted into a mouse vascularised chamber model. This in vivo xenograft model will provide the basis of future studies of LM biology and testing of potential pharmacological interventions for patients with lymphatic malformations.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Brice G, Child AH, Evans A, Bell R, Mansour S, Burnand K, Sarfarazi M, Jeffery S, Mortimer P (2005) Milroy disease and the VEGFR-3 mutation phenotype. J Med Genet 42:98–102

    Article  CAS  PubMed  Google Scholar 

  2. Nakamura K, Rockson SG (2007) Biomarkers of lymphatic function and disease: state of the art and future directions. Mol Diagn Ther 11:227–238

    Article  CAS  PubMed  Google Scholar 

  3. Castro EC, Galambos C (2009) Prox-1 and VEGFR3 antibodies are superior to D2-40 in identifying endothelial cells of lymphatic malformations-a proposal of a new immunohistochemical panel to differentiate lymphatic from other vascular malformations. Pediatr Dev Pathol 12:187–194

    Article  PubMed  Google Scholar 

  4. Filston HC (1994) Hemangiomas, cystic hygromas, and teratomas of the head and neck. Semin Pediatr Surg 3:147–159

    CAS  PubMed  Google Scholar 

  5. Rottem S, Bronshtein M (1990) Transvaginal sonographic diagnosis of congenital anomalies between 9 weeks and 16 weeks menstrual age. J Clin Ultrasound 18:307–314

    Article  CAS  PubMed  Google Scholar 

  6. Puig S, Casati B, Staudenherz A, Paya K (2005) Vascular low-flow malformations in children: current concepts for classification, diagnosis and therapy. Eur J Radiol 53:35–45

    Article  PubMed  Google Scholar 

  7. Mulliken JB, Glowacki J (1982) Hemangiomas and vascular malformations in infants and children: a classification based on endothelial characteristics. Plast Reconstr Surg 69:412–422

    Article  CAS  PubMed  Google Scholar 

  8. Enjolras O, Wassef M, Chapot R (2007) Colour atlas of vascular tumours and vascular malformations. Cambridge University Press, New York

    Book  Google Scholar 

  9. Vikkula M, Boon LM, Mulliken JB, Olsen BR (1998) Molecular basis of vascular anomalies. Trends Cardiovasc Med 8:281–292

    Article  CAS  PubMed  Google Scholar 

  10. Padwa BL, Hayward PG, Ferraro NF, Mulliken JB (1995) Cervicofacial lymphatic malformation: clinical course, surgical intervention, and pathogenesis of skeletal hypertrophy. Plast Reconstr Surg 95:951–960

    Article  CAS  PubMed  Google Scholar 

  11. Whimster IW (1976) The pathology of lymphangioma circumscriptum. Br J Dermatol 94:473–486

    Article  CAS  PubMed  Google Scholar 

  12. Mulliken JB, Glowacki J (1982) Classification of pediatric vascular lesions. Plast Reconstr Surg 70:120–121

    Article  CAS  PubMed  Google Scholar 

  13. Galambos C, Nodit L (2005) Identification of lymphatic endothelium in pediatric vascular tumors and malformations. Pediatr Dev Pathol 8:181–189

    Article  PubMed  Google Scholar 

  14. Edwards PD, Rahbar R, Ferraro NF, Burrows PE, Mulliken JB (2005) Lymphatic malformation of the lingual base and oral floor. Plast Reconstr Surg 115:1906–1915

    Article  CAS  PubMed  Google Scholar 

  15. Brouillard P, Vikkula M (2003) Vascular malformations: localized defects in vascular morphogenesis. Clin Genet 63:340–351

    Article  CAS  PubMed  Google Scholar 

  16. Huang H-Y, Ho C-C, Huang P-H, Hsu S-M (2001) Co-Expression of VEGF-C and its receptors, VEGFR-2 and VEGFR-3, in endothelial cells of lymphangioma. Implication in autocrine or paracrine regulation of lymphangioma. Lab Invest 81:1729–1734

    Article  CAS  PubMed  Google Scholar 

  17. Jackson IT, Carreno R, Potparic Z, Hussain K (1993) Hemangiomas, vascular malformations, and lymphovenous malformations: classification and methods of treatment. Plast Reconstr Surg 91:1216–1230

    Article  CAS  PubMed  Google Scholar 

  18. Hitchcock T, Niklason L (2008) Lymphatic tissue engineering: progress and prospects. Ann N Y Acad Sci 1131:44–49

    Article  PubMed Central  PubMed  Google Scholar 

  19. Lokmic Z, Mitchell GM (2008) Engineering the microcirculation. Tissue Eng Part B Rev 14:87–103

    Article  CAS  PubMed  Google Scholar 

  20. Cronin KJ, Messina A, Knight KR, Cooper-White JJ, Stevens GW, Penington AJ, Morrison WA (2004) New murine model of spontaneous autologous tissue engineering, combining an arteriovenous pedicle with matrix materials. Plast Reconstr Surg 113:260–269

    Article  PubMed  Google Scholar 

  21. Tilkorn DJ, Davies EM, Keramidaris E, Dingle AM, Gerrand Y-W, Taylor CJ, Han X-L, Palmer JA, Penington AJ, Mitchell CA, Morrison WA, Dusting GJ, Mitchell GM (2012) The in vitro preconditioning of myoblasts to enhance subsequent survival in an in vivo tissue engineering chamber model. Biomaterials 33:3868–3879

    Article  CAS  PubMed  Google Scholar 

  22. Alitalo K, Tammela T, Petrova TV (2005) Lymphangiogenesis in development and human disease. Nature 438:946–953

    Article  CAS  PubMed  Google Scholar 

  23. Zeng Y, Opeskin K, Horvath LG, Sutherland RL, Williams ED (2005) Lymphatic vessel density and lymph node metastasis in prostate cancer. Prostate 65:222–230

    Article  CAS  PubMed  Google Scholar 

  24. Zeng Y, Wang F, Williams ED, Chow CW (2005) Lymphatics in the alimentary tract of children in health and disease: study on mucosal biopsies using the monoclonal antibody d2-40. Pediatr Dev Pathol 8:541–549

    Article  CAS  PubMed  Google Scholar 

  25. Zeng Y, Opeskin K, Goad J, Williams ED (2006) Tumor-induced activation of lymphatic endothelial cells via vascular endothelial growth factor receptor-2 is critical for prostate cancer lymphatic metastasis. Cancer Res 66:9566–9575

    Article  CAS  PubMed  Google Scholar 

  26. Coso S, Zeng Y, Opeskin K, Williams ED (2012) Vascular endothelial growth factor receptor-3 directly interacts with phosphatidylinositol 3-kinase to regulate lymphangiogenesis. PLoS ONE 7(6):e39558. doi:10.1371/journal.pone.0039558

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Louis KS, Siegel AC (2011) Cell viability analysis using trypan blue: manual and automated methods. Methods Mol Biol 740:7–12

  28. Howard CV, Reed MG (2005) Unbiased stereology. BIOS Scientific Publishers, Taylor & Francis Group, Oxon

    Google Scholar 

  29. Wilting J, Papoutsi M, Christ B, Nicolaides KH, von Kaisenberg CS, Borges J, Stark GB, Alitalo K, Tomarev SI, Niemeyer C, Rössler J (2002) The transcription factor Prox1 is a marker for lymphatic endothelial cells in normal and diseased human tissues. FASEB J 16:1271–1273

    CAS  PubMed  Google Scholar 

  30. Corselli M, Chen CW, Sun B, Yap S, Rubin JP, Peault B (2012) The tunica adventitia of human arteries and veins as a source of mesenchymal stem cells. Stem Cells Dev 21:1299–1308

    Article  CAS  PubMed  Google Scholar 

  31. Swetman GL, Berk DR, Vasanawala SS, Feinstein JA, Lane AT, Bruckner AL (2012) Sildenafil for severe lymphatic malformations. N Engl J Med 366:384–386

    Article  CAS  PubMed  Google Scholar 

  32. Dai Y, Hou F, Saad A, Fan CY, Buckmiller LM, Suen JY, Richter GT (2011) Preliminary investigation of human lymphatic malformations in vitro. Laryngoscope 121:2435–2442

    Article  PubMed  Google Scholar 

  33. Sun Y, Jia J, Zhang W, Liu B, Zhang Z, Zhao Y (2011) A reproducible in vivo model of lymphatic malformation in rats. J Comp Pathol 145:390–398

    Article  CAS  PubMed  Google Scholar 

  34. Bennett G, April M, Huvos AG (2004) Lymphatic malformation of the sphenoid sinus in a pediatric patient. Otolaryngol Head Neck Surg 131:784–786

    Article  PubMed  Google Scholar 

  35. Zhang XD, Gillespie SK, Hersey P (2004) Staurosporine induces apoptosis of melanoma by both caspase-dependent and caspase-independent apoptotic pathways. Mol Cancer Ther 2004(3):187–197

    Google Scholar 

  36. Cavallaro U, Dejana E (2011) Adhesion molecule signalling: not always a sticky business. Nat Rev Mol Cell Biol 12:189–197

    Google Scholar 

  37. Dejana E (2004) Endothelial cell–cell junctions: happy together. Nat Rev Mol Cell Biol 5:261–270

    Article  CAS  PubMed  Google Scholar 

  38. Raymond MA, Vigneault N, Luyckx V, Hebert MJ (2002) Paracrine repercussions of preconditioning on angiogenesis and apoptosis of endothelial cells. Biochem Biophys Res Commun 291:261–269

    Article  CAS  PubMed  Google Scholar 

  39. Wautier MP, Boval B, Chappey O, Enjolras O, Wernert N, Merland JJ, Wautier JL (1999) Cultured endothelial cells from human arteriovenous malformations have defective growth regulation. Blood 94:2020–2028

    CAS  PubMed  Google Scholar 

  40. Egeblad M, Nakasone ES, Werb Z (2010) Tumors as organs: complex tissues that interface with the entire organism. Dev Cell 18:884–901

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  41. Makinen T, Adams RH, Bailey J, Lu Q, Ziemiecki A, Alitalo K, Klein R, Wilkinson GA (2005) PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature. Genes Dev 19:397–410

    Article  PubMed  Google Scholar 

  42. Pflicke H, Sixt M (2009) Preformed portals facilitate dendritic cell entry into afferent lymphatic vessels. J Exp Med 206:2925–2935

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  43. Mikhaylova M, Mori N, Wildes FB, Walczak P, Gimi B, Bhujwalla ZM (2008) Hypoxia increases breast cancer cell-induced lymphatic endothelial cell migration. Neoplasia 10:380–389

    CAS  PubMed Central  PubMed  Google Scholar 

  44. Garaffa E, Caprara V, Di Castro V, Rosano L, Bagnato A, Spinella F (2012) Endothelin-1 cooperates with hypoxia to induce vascular-like structures through vascular endothelial growth factors -C, -D and -A in lymphatic endothelial cells. Life Sci 19:638–643

    Article  Google Scholar 

  45. Irigoyen M, Anso E, Martinez E, Garoya M, Martinez-Irujo JJ, Rouzant A (2007) Hypoxia alters the adhesive properties of lymphatic endothelial cells. A transcriptional and functional study. Biochim Biophys Acta 1773:880–890

    Article  CAS  PubMed  Google Scholar 

  46. Williams SP, Karnezis T, Achen MG, Stacker SA (2010) Targeting lymphatic vessel functions through tyrosine kinases. J Angiogenes Res 2:13

    Article  PubMed Central  PubMed  Google Scholar 

  47. Folkman J, Kaipainen A (2004) Genes tell lymphatics to sprout or not. Nat Immunol 5:11–12

    Article  CAS  PubMed  Google Scholar 

  48. Norgall S, Papoutsi M, Rossler J, Schweigerer L, Wilting J, Weich HA (2007) Elevated expression of VEGFR-3 in lymphatic endothelial cells from lymphangiomas. BMC Cancer 7:105–122

    Article  PubMed Central  PubMed  Google Scholar 

  49. Stacker S, Hughes R, Williams R, Achen M (2006) Current strategies for modulating lymphangiogenesis signalling pathways in human disease. Curr Med Chem 13:783–792

    Article  CAS  PubMed  Google Scholar 

  50. Dixelius J, Makinen T, Wirzenius M, Karkkainen MJ, Wernstedt C, Alitalo K, Claesson-Welsh L (2003) Ligand-induced vascular endothelial growth factor receptor-3 (VEGFR-3) heterodimerization with VEGFR-2 in primary lymphatic endothelial cells regulates tyrosine phosphorylation sites. J Biol Chem 278:40973–40979

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Michelle Marchionna for technical assistance in the early stages of this project. This work was supported by grants from The National Health and Medical Research Council (NHMRC #566886), The Hector Waldron Pride Charitable Trust (EDW, AJP) and The William Buckland Foundation (AJP, MM). EDW was supported by an NHMRC Career Development Award (#519539). The O’Brien Institute and Monash Institute of Medical Research acknowledge support from the Victorian State Government’s Department of Innovation, Industry and Regional Development’s Operational Infrastructure Support Program.

Conflict of interest

There are no conflicts of interests to declare.

Author information

Author notes

  1. Nicholas Koh Wee Chong

    Present address: Department of Pathology, Tan Tock Seng Hospital, 11 Jalan Tan Tock Seng, Singapore, Singapore

Authors and Affiliations

  1. O’Brien Institute, 42 Fitzroy Street, Fitzroy, VIC, 3065, Australia

    Zerina Lokmic, Geraldine M. Mitchell, Nicholas Koh Wee Chong, Jacqueline Bastiaanse, Yi-Wen Gerrand, Yiping Zeng & Anthony J. Penington

  2. Department of Surgery, St Vincent’s Hospital, The University of Melbourne, 29 Regent Street, Fitzroy, VIC, 3065, Australia

    Zerina Lokmic, Geraldine M. Mitchell, Nicholas Koh Wee Chong, Elizabeth D. Williams & Anthony J. Penington

  3. Murdoch Childrens Research Institute and The Royal Children’s Hospital, Flemington Rd, Parkville, VIC, 3052, Australia

    Zerina Lokmic & Anthony J. Penington

  4. Faculty of Health Sciences, The Australian Catholic University, 115 Victoria Pde, Fitzroy, VIC, 3065, Australia

    Geraldine M. Mitchell & Yi-Wen Gerrand

  5. Centre for Cancer Research, Monash Institute of Medical Research, Monash University, 27-31 Wright St., Clayton, VIC, 3168, Australia

    Elizabeth D. Williams

Authors
  1. Zerina Lokmic
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Geraldine M. Mitchell
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nicholas Koh Wee Chong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jacqueline Bastiaanse
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yi-Wen Gerrand
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yiping Zeng
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Elizabeth D. Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Anthony J. Penington
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zerina Lokmic.

Electronic supplementary material

Below is the link to the electronic supplementary material.

10456_2013_9371_MOESM1_ESM.tif

Expression of CD34 in the lymphatic malformation human tissue. A CD34 expression is not detected in the lymphatic malformation endothelium (#). CD34 is present on the stromal cells in the connective tissue, in cells residing in tunica adventitia of blood vessels and in the endothelium of blood vessels (arrow). B The absence of CD34 from the lymphatic malformation (#) was further confirmed with double labelling of CD34 and CD31 where co-expression of the two is found in blood vessels only (arrow) (TIFF 40192 kb)

10456_2013_9371_MOESM2_ESM.tif

Deposition of connective tissue in the LM xenograft model. Masson’s trichrome stain labels connective tissue component collagen blue (arrows). A, B Connective tissue in 1 week LEC-implanted mouse chamber at low and high magnification respectively. C, D Connective and adipose tissue in 4-week LEC implanted chambers low and high magnification respectively. Note that collagen is most evident at tissue periphery. E, F Connective tissue in 1 week LM-LEC implanted mouse chamber low and high magnification respectively. G, H Connective and adipose tissue in 4-week LM-LEC implanted chambers at low and high magnification respectively. Arrows point to connective tissue in the vicinity of LM vessels (≠). Host artery (#) and vein (*) are present in the chambers (TIFF 50945 kb)

10456_2013_9371_MOESM3_ESM.tif

Cell Proliferation in LM-LEC implanted chambers. A Ki-67 positive proliferating cells were identified in human lymph node, positive control tissue. B, C, D Rare Ki-67-labelled cells were identified in lymphatic malformation vessels at 4 weeks (*). E Ki67 labelled cells were also identified in the host tissue and F in the vicinity of the LM vessels (*). Note variable degree of Ki-67 staining indicative of cell entry or exit from cell proliferation cycle (TIFF 26008 kb)

10456_2013_9371_MOESM4_ESM.tif

Cell death in LM-LEC implanted chambers. A Cleaved caspase-3 positive cells (arrowheads) were identified in human lymph node, positive control tissue. Note different staining patterns within the lymph node follicle. B Cleaved caspase-3 positive cells (arrowheads) were present at the edge of the chamber tissue at 4 weeks and C in the developing host tissue in 1 and 2-week old chambers. D, E Both LEC and LM-LEC implanted chambers contain dead LEC and LM-LECs in the Matrigel where weak residual caspase-3 staining was observed (arrowheads). Note refractive brown dynabead® in the tissue section shown in E. F Hematoxylin stained apoptotic bodies (arrowheads) were evident in both the LEC and LM-LEC implanted chambers in haematoxylin-eosin stained tissues (TIFF 25329 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lokmic, Z., Mitchell, G.M., Koh Wee Chong, N. et al. Isolation of human lymphatic malformation endothelial cells, their in vitro characterization and in vivo survival in a mouse xenograft model. Angiogenesis 17, 1–15 (2014). https://doi.org/10.1007/s10456-013-9371-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10456-013-9371-8

Keywords

Search

Navigation