, Volume 9, Issue 4, pp 209–224 | Cite as

Unique vascular phenotypes following over-expression of individual VEGFA isoforms from the developing lens

  • Christopher A. Mitchell
  • Catrin S. Rutland
  • Michael Walker
  • Muneeb Nasir
  • Alexander J. E. Foss
  • Christine Stewart
  • Holger Gerhardt
  • Moritz A. Konerding
  • Werner Risau
  • Hannes C. A. Drexler
Original Paper


Formation of a correctly organised vasculature and subsequently embryonic survival is critically dependent on the dosage and site-specific expression of VEGF. Murine VEGF exists in three common isoforms (viz. 120, 164 and 188 amino acids) having different organ specific distribution levels. Gene knock-in studies show that expression of any of the individual isoforms of VEGF extends survival until birth, although each is associated with distinct organ-specific abnormalities. Comparison of the effects of VEGF isoform expression is complicated by the general lethality of mis-expression, in addition to cumulative effects of adjacent tissues from the inappropriately patterned vasculature. Here we investigate the effects of over-expression of individual VEGFA isoforms from the lens-specific αA-Crystallin promoter and characterise their effects on the vessel morphology of the hyaloid and developing retinal vasculature. Since the hyaloid vasculature is an anatomically distinct, transient vasculature of the eye, comprising 3 cell types (endothelium, pericytes and macrophages) it is possible to more readily interpret the role of individual VEGF-A isoforms in vascular pattern formation in this model. The severity of the vascular phenotype, characterised by a hyperplastic hyaloid at E13.5 and subsequently retinal vascular patterning and ocular defects, is most severe in transgenics over-expressing the more diffusible forms of VEGFA (120 and 164), whereas in VEGFA188 transgenics the hyaloid vascular defects partially resolve post-natally. The results of this study indicate that individual isoforms of VEGFA induce distinct vascular phenotypes in the eye during embryonic development and that their relative doses provide instructive cues for vascular patterning.


Mouse Transgenic Lens Angiogenesis VEGF Isoforms 



The authors wish to thank Mrs. M. Mitchell for expert animal care, Dr. Karim Bakri for sample processing, as well as Mr. Trevor Gray and Phillip Hinson for assistance with electron microscopy. We thank G. Breier for VEGF isoform plasmids, P. Overbeek for the CPV2 plasmid as well as L.A.G. Lucas and N.J. Duffin for assistance with microscopy. H. Gerhardt was supported by an EMBO fellowship.


  1. 1.
    Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawling J, Moons L, Collen D, Risau W, Nagy A (1996) Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380:435–439CrossRefPubMedGoogle Scholar
  2. 2.
    Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O’Shea KS, Powell-Braxton L, Hillan KJ, Moore MW (1996) Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 380:439–442CrossRefPubMedGoogle Scholar
  3. 3.
    Haigh JJ, Gerber HP, Ferrara N, Wagner EF (2000) Conditional inactivation of VEGF-A in areas of collagen2a1 expression results in embryonic lethality in the heterozygous state. Development 127:1445–1453PubMedGoogle Scholar
  4. 4.
    Gerber HP, Hillan KJ, Ryan AM, Kowalski J, Keller GA, Rangell L, Wright BD, Radtke F, Aguet M, Ferrara N (1999) VEGF is required for growth and survival in neonatal mice. Development 126:1149–1159PubMedGoogle Scholar
  5. 5.
    Ferrara N, Houck K, Jakeman L, Leung DW (1992) Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr Rev 13:18–32CrossRefPubMedGoogle Scholar
  6. 6.
    Shima DT, Kuroki M, Deutsch U, Ng YS, Adamis AP, D’Amore PA (1996) The mouse gene for vascular endothelial growth factor. Genomic structure, definition of the transcriptional unit, and characterization of transcriptional and post-transcriptional regulatory sequences. J Biol Chem 271:3877–3883CrossRefPubMedGoogle Scholar
  7. 7.
    Ng YS, Rohan R, Sunday ME, Demello DE, D’Amore PA (2001) Differential expression of VEGF isoforms in mouse during development and in the adult. Dev Dynam 220:112–121CrossRefGoogle Scholar
  8. 8.
    Park JE, Keller GA, Ferrara N (1993) The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF. Mol Biol Cell 4:1317–1326PubMedGoogle Scholar
  9. 9.
    Keyt BA, Berleau LT, Nguyen HV, Chen H, Heinsohn H, Vandlen R, Ferrara N (1996) The carboxyl-terminal domain (111–165) of vascular endothelial growth factor is critical for its mitogenic potency. J Biol Chem 271:7788–7795CrossRefPubMedGoogle Scholar
  10. 10.
    Forsten KE, Fannon M, Nugent MA (2000) Potential mechanisms for the regulation of growth factor binding by heparin. J Theor Biol 205:215–230CrossRefPubMedGoogle Scholar
  11. 11.
    Carmeliet P, Ng YS, Nuyens D, Theilmeier G, Brusselmans K, Cornelissen I, Ehler E, Kakkar VV, Stalmans I, Mattot V, Perriard JC, Dewerchin M, Flameng W, Nagy A, Lupu F, Moons L, Collen D, Damore PA, Shima DT (1999) Impaired myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the vascular endothelial growth factor isoforms VEGF(164) and VEGF(188). Nat Med 5:495–502CrossRefPubMedGoogle Scholar
  12. 12.
    Stalmans I, Ng YS, Rohan R, Fruttiger M, Bouche A, Yuce A, Fujisawa H, Hermans B, Shani M, Jansen S, Hicklin D, Anderson DJ, Gardiner T, Hammes HP, Moons L, Dewerchin M, Collen D, Carmeliet P, D’Amore PA (2002) Arteriolar and venular patterning in retinas of mice selectively expressing VEGF isoforms. J Clin Invest 109:327–336CrossRefPubMedGoogle Scholar
  13. 13.
    Maes C, Carmeliet P, Moermans K, Stockmans I, Smets N, Collen D, Bouillon R, Carmeliet G (2002) Impaired angiogenesis and endochondral bone formation in mice lacking the vascular endothelial growth factor isoforms VEGF(164) and VEGF(188). Mech Dev 111:61–73CrossRefPubMedGoogle Scholar
  14. 14.
    Overbeek PA, Chepelinsky AB, Khillan JS, Piatigorsky J, Westphal H (1985) Lens-specific expression and developmental regulation of the bacterial chloramphenicol acetyltransferase gene driven by the murine alpha A-crystallin promoter in transgenic mice. Proc Natl Acad Sci USA 82:7815–7819CrossRefPubMedGoogle Scholar
  15. 15.
    Ash JD, Overbeek PA (2000) Lens-specific VEGF-A expression induces angioblast migration and proliferation and stimulates angiogenic remodeling. Dev Biol 223:383–398CrossRefPubMedGoogle Scholar
  16. 16.
    Mitchell CA, Risau W, Drexler HCA (1998) Regression of vessels in the tunica vasculosa lentis is initiated by coordinated endothelial apoptosis: a role for vascular endothelial growth factor as a survival factor for endothelium. Dev Dynam 213:322–333CrossRefGoogle Scholar
  17. 17.
    Reneker LW, Silversides DW, Patel K, Overbeek PA (1995) TGF alpha can act as a chemoattractant to perioptic mesenchymal cells in developing mouse eyes. Development 121:1669–1680PubMedGoogle Scholar
  18. 18.
    Hovey RC, Goldhar AS, Baffi J, Vonderhaar BK (2001) Transcriptional regulation of vascular endothelial growth factor expression in epithelial and stromal cells during mouse mammary gland development. Mol Endocrinol 15:819–831CrossRefPubMedGoogle Scholar
  19. 19.
    Marti HJ, Bernaudin M, Bellail A, Schoch H, Euler M, Petit E, Risau W (2000) Hypoxia-induced vascular endothelial growth factor expression precedes neovascularization after cerebral ischemia. Am J Pathol 156:965–976PubMedGoogle Scholar
  20. 20.
    Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, Jeltsch M, Mitchell C, Alitalo K, Shima D, Betsholtz C (2003) VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 161:1163–1177CrossRefPubMedGoogle Scholar
  21. 21.
    Kretzler M, Schroppel B, Merkle M, Huber S, Mundel P, Horster M, Schlondorff D (1998) Detection of multiple vascular endothelial growth factor splice isoforms in single glomerular podocytes. Kidney Int 54:S159–S161CrossRefGoogle Scholar
  22. 22.
    Halder JB, Zhao X, Soker S, Paria BC, Klagsbrun M, Das SK, Dey SK (2000) Differential expression of VEGF isoforms and VEGF(164)-specific receptor neuropilin-1 in the mouse uterus suggests a role for VEGF(164) in vascular permeability and angiogenesis during implantation. Genesis 26:213–224CrossRefPubMedGoogle Scholar
  23. 23.
    Krussel JS, Behr B, Milki AA, Hirchenhain J, Wen Y, Bielfeld P, Lake Polan M (2001) Vascular endothelial growth factor (VEGF) mRNA splice variants are differentially expressed in human blastocysts. Mol Hum Reprod 7:57–63CrossRefPubMedGoogle Scholar
  24. 24.
    Simon M, Grone HJ, Johren O, Kullmer J, Plate KH, Risau W, Fuchs E (1995) Expression of vascular endothelial growth-factor and its receptors in human renal ontogeny and in adult kidney. Am J Physiol Renal Fluid Electrolyte Physiol 37:F240–F250Google Scholar
  25. 25.
    Cheung N, Wong MP, Yuen ST, Leung SY, Chung LP (1998) Tissue-specific expression pattern of vascular endothelial growth factor isoforms in the malignant transformation of lung and colon. Hum Pathol 29:910–914CrossRefPubMedGoogle Scholar
  26. 26.
    Okamoto K, Oshika Y, Fukushima Y, Ohnishi Y, Tokunaga T, Tomii Y, Kijima H, Yamazaki H, Ueyama Y, Tamaoki N, Nakumura M (1999) Xenografts of human solid tumors frequently express cellular-associated isoform of vascular endothelial growth factor (VEGF) 189. Oncol Rep 6:1201–1204PubMedGoogle Scholar
  27. 27.
    Grunstein J, Masbad JJ, Hickey R, Giordano F, Johnson RS (2000) Isoforms of vascular endothelial growth factor act in a coordinate fashion to recruit and expand tumor vasculature. Mol Cell Biol 20:7282–7291CrossRefPubMedGoogle Scholar
  28. 28.
    Yu JL, Rak JW, Klement G, Kerbel RS (2002) Vascular endothelial growth factor isoform expression as a determinant of blood vessel patterning in human melanoma xenografts. Cancer Res 62:1838–1846PubMedGoogle Scholar
  29. 29.
    Tober KL, Cannon RE, Spalding JW, Oberyszyn TM, Parrett ML, Rackoff AI, Oberyszyn AS, Tennant RW, Robertson FM (1998) Comparative expression of novel vascular endothelial growth factor vascular permeability factor transcripts in skin, papillomas, and carcinomas of v-Ha-ras Tg.AC transgenic mice and FVB/N mice [Full text delivery]. Biochem Biophys Res Commun 247:644–653CrossRefPubMedGoogle Scholar
  30. 30.
    Mann IC (1964) The development of the human eye. Grune and Stratton Inc., New YorkGoogle Scholar
  31. 31.
    Balazs EA, Toth LZ, Ozanics V (1980) Cytological studies on the developing vitreous as related to the hyaloid vessel system. Albrecht Von Graefes Archiv fur Klinische und Experimentelle Ophthalmologie 213:71–85CrossRefPubMedGoogle Scholar
  32. 32.
    Lang R (1997) Apoptosis in mammalian eye development: lens morphogenesis, vascular regression and immune privilege. Cell Death Differ 4:12–20CrossRefPubMedGoogle Scholar
  33. 33.
    de Iongh R, McAvoy JW (1992) Distribution of acidic and basic fibroblast growth factors (FGF) in the foetal rat eye: implications for lens development. Growth Factors 6:159–177PubMedCrossRefGoogle Scholar
  34. 34.
    Parmigiani CM, McAvoy JW (1989) A morphometric analysis of the development of the rat lens capsule. Curr Eye Res 8:1271–1277PubMedGoogle Scholar
  35. 35.
    Davies MJ, Mitchell CA, Maley MAL, Grounds MD, Harvey AR, Plant GW, Wood DJ, Hong Y, Chirila TV (1997) In vitro assessment of the biological activity of basic fibroblast growth factor released from various polymers and biomatrices. J Biomater Appl 12:31–56Google Scholar
  36. 36.
    Mudhar HS, Pollock RA, Wang C, Stiles CD, Richardson WD (1993) PDGF and its receptors in the developing rodent retina and optic nerve. Development 118:539–552PubMedGoogle Scholar
  37. 37.
    Robinson ML, Overbeek PA, Verran DJ, Grizzle WE, Stockard CR, Friesel R, Maciag T, Thompson JA (1995) Extracellular FGF-1 acts as a lens differentiation factor in transgenic mice. Development 121:505–514PubMedGoogle Scholar
  38. 38.
    Lovicu FJ, Overbeek PA (1998) Overlapping effects of different members of the FGF family on lens fiber differentiation in transgenic mice. Development 125:3365–3377PubMedGoogle Scholar
  39. 39.
    Reneker LW, Overbeek PA (1996) Lens-specific expression of PDGF-A alters lens growth and development. Dev Biol 180:554–565CrossRefPubMedGoogle Scholar
  40. 40.
    Cleaver O, Krieg PA (1998) VEGF mediates angioblast migration during development of the dorsal aorta in Xenopus. Development 125:3905–3914PubMedGoogle Scholar
  41. 41.
    Shoji W, Isogai S, Sato-Maeda M, Obinata M, Kuwada JY (2003) Semaphorin3a1 regulates angioblast migration and vascular development in zebrafish embryos. Development 130:3227–3236CrossRefPubMedGoogle Scholar
  42. 42.
    Ruhrberg C, Gerhardt H, Golding M, Watson R, Ioannidou S, Fujisawa H, Betsholtz C, Shima DT (2002) Spatially restricted patterning cues provided by heparin-binding VEGF-A control blood vessel branching morphogenesis. Genes Dev 16:2684–2698CrossRefPubMedGoogle Scholar
  43. 43.
    Wilting J, Birkenhager R, Eichmann A, Kurz H, Martiny-Baron G, Marme D, McCarthy JE, Christ B, Weich HA (1996) VEGF121 induces proliferation of vascular endothelial cells and expression of flk-1 without affecting lymphatic vessels of chorioallantoic membrane. Dev Biol 176:76–85CrossRefPubMedGoogle Scholar
  44. 44.
    Drake CJ, Little CD (1995) Exogenous vascular endothelial growth factor induces malformed and hyperfused vessels during embryonic neovascularization. Proc Natl Acad Sci USA 92:7657–7661CrossRefPubMedGoogle Scholar
  45. 45.
    Cheng SY, Nagane M, Huang HJS, Cavenee WK (1997) Intracerebral tumor-associated hemorrhage caused by overexpression of the vascular endothelial growth factor isoforms VEGF(121) and VEGF(165) but not VEGF(189). Proc Natl Acad Sci USA 94:12081–12087CrossRefPubMedGoogle Scholar
  46. 46.
    Dor Y, Djonov V, Abramovitch R, Itin A, Fishman GI, Carmeliet P, Goelman G, Keshet E (2002) Conditional switching of VEGF provides new insights into adult neovascularization and pro-angiogenic therapy. EMBO J 21:1939–1947CrossRefPubMedGoogle Scholar
  47. 47.
    Pettersson A, Nagy JA, Brown LF, Sundberg C, Morgan E, Jungles S, Carter R, Krieger JE, Manseau EJ, Harvey VS, Eckelhoefer IA, Feng D, Dvorak AM, Mulligan RC, Dvorak HF (2000) Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular permeability factor/vascular endothelial growth factor. Lab Invest 80:99–115PubMedCrossRefGoogle Scholar
  48. 48.
    Beck L Jr, D’Amore PA (1997) Vascular development: cellular and molecular regulation. Faseb J 11:365–373PubMedGoogle Scholar
  49. 49.
    Ishida A, Murray J, Saito Y, Kanthou C, Benzakour O, Shibuya M, Wijelath ES (2001) Expression of vascular endothelial growth factor receptors in smooth muscle cells. J Cell Physiol 188:359–368CrossRefPubMedGoogle Scholar
  50. 50.
    Yamagishi S, Yonekura H, Yamamoto Y, Fujimori H, Sakurai S, Tanaka N, Yamamoto H (1999) Vascular endothelial growth factor acts as a pericyte mitogen under hypoxic conditions. Lab Invest 79:501–509PubMedGoogle Scholar
  51. 51.
    Benjamin LE, Hemo I, Keshet E (1998) A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF- B and VEGF. Development 125:1591–1598PubMedGoogle Scholar
  52. 52.
    Hirschi KK, Rohovsky SA, Beck LH, Smith SR, D’Amore PA (1999) Endothelial cells modulate the proliferation of mural cell precursors via platelet-derived growth factor-BB and heterotypic cell contact. Circ Res 84:298–305PubMedGoogle Scholar
  53. 53.
    Lindahl P, Hellstrom M, Kalen M, Betsholtz C (1998) Endothelial-perivascular cell signaling in vascular development: lessons from knockout mice. Curr Opin Lipidol 9:407–411CrossRefPubMedGoogle Scholar
  54. 54.
    Lindblom P, Gerhardt H, Liebner S, Abramsson A, Enge M, Hellstrom M, Backstrom G, Fredriksson S, Landegren U, Nystrom HC, Bergstrom G, Dejana E, Ostman A, Lindahl P, Betsholtz C (2003) Endothelial PDGF-B retention is required for proper investment of pericytes in the microvessel wall. Genes Dev 17:1835–1840CrossRefPubMedGoogle Scholar
  55. 55.
    Diez-Roux G, Lang RA. (1997) Macrophages induce apoptosis in normal cells in vivo. Development 124:3633–3638PubMedGoogle Scholar
  56. 56.
    Diez-Roux G, Argilla M, Makarenkova H, Ko K, Lang RA (1999) Macrophages kill capillary cells in G1 phase of the cell cycle during programmed vascular regression. Development 126:2141–2147PubMedGoogle Scholar
  57. 57.
    Lobov IB, Rao S, Carroll TJ, Vallance JE, Ito M, Ondr JK, Kurup S, Glass DA, Patel MS, Shu W, Morrisey EE, McMahon AP, Karsenty G, Lang RA (2005) WNT7b mediates macrophage-induced programmed cell death in patterning of the vasculature. Nature 437:417–421CrossRefPubMedGoogle Scholar
  58. 58.
    Fruttiger M (2002) Development of the mouse retinal vasculature: angiogenesis versus vasculogenesis. Invest Ophthalmol Vis Sci 43:522–527PubMedGoogle Scholar
  59. 59.
    Stone J, Itin A, Alon T, Pe’er J, Gnessin H, Chan-Ling T, Keshet E (1995) Development of retinal vasculature is mediated by hypoxia-induced vascular endothelial growth factor (VEGF) expression by neuroglia. J Neurosci 15:4738–4747PubMedGoogle Scholar
  60. 60.
    Klagsbrun M, Takashima S, Mamluk R (2002) The role of neuropilin in vascular and tumor biology. Adv Exp Med Biol 515:33–48PubMedGoogle Scholar
  61. 61.
    Soker S, Takashima S, Miao HQ, Neufeld G, Klagsbrun M (1998) Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 92:735–745CrossRefPubMedGoogle Scholar
  62. 62.
    Ishihama H, Ohbayashi M, Kurosawa N, Kitsukawa T, Matsuura O, Miyake Y, Muramatsu T (2001) Colocalization of neuropilin-1 and Flk-1 in retinal neovascularization in a mouse model of retinopathy. Invest Ophthalmol Vis Sci 42:1172–1178PubMedGoogle Scholar
  63. 63.
    Plouet J, Moro F, Bertagnolli S, Coldeboeuf N, Mazarguil H, Clamens S, Bayard F (1997) Extracellular cleavage of the vascular endothelial growth factor 189-amino acid form by urokinase is required for its mitogenic effect. J Biol Chem 272:13390–13396CrossRefPubMedGoogle Scholar
  64. 64.
    Ogata N, Yamanaka R, Yamamoto C, Miyashiro M, Kimoto T, Takahashi K, Maruyama K, Uyama M (1998) Expression of vascular endothelial growth factor and its receptor, KDR, following retinal ischemia-reperfusion injury in the rat. Curr Eye Res 17:1087–1096CrossRefPubMedGoogle Scholar
  65. 65.
    Stout AU, Stout JT (2003) Retinopathy of prematurity. Pediatr Clin North Am 50:77–87, viGoogle Scholar
  66. 66.
    Alon T, Hemo I, Itin A, Pe’er J, Stone J, Keshet E (1995) Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat Med 1:1024–1028CrossRefPubMedGoogle Scholar
  67. 67.
    Zhang HT, Scott PA, Morbidelli L, Peak S, Moore J, Turley H, Harris AL, Ziche M, Bicknell R (2000) The 121 amino acid isoform of vascular endothelial growth factor is more strongly tumorigenic than other splice variants in vivo. Br J Cancer 83:63–68CrossRefPubMedGoogle Scholar
  68. 68.
    Silbert M, Gurwood AS (2000) Persistent hyperplastic primary vitreous. Clin Eye Vis Care 12:131–137CrossRefPubMedGoogle Scholar
  69. 69.
    Mullner-Eidenbock A, Amon M, Moser E, Klebermass N (2004) Persistent fetal vasculature and minimal fetal vascular remnants: a frequent cause of unilateral congenital cataracts. Ophthalmology 111:906–913CrossRefPubMedGoogle Scholar
  70. 70.
    Campochiaro PA (2000) Retinal and choroidal neovascularization. J Cell Physiol 184:301–310CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science + Business Media B.V. 2006

Authors and Affiliations

  • Christopher A. Mitchell
    • 1
    • 2
    • 3
  • Catrin S. Rutland
    • 1
  • Michael Walker
    • 2
  • Muneeb Nasir
    • 1
  • Alexander J. E. Foss
    • 4
  • Christine Stewart
    • 3
  • Holger Gerhardt
    • 5
  • Moritz A. Konerding
    • 6
  • Werner Risau
    • 2
  • Hannes C. A. Drexler
    • 2
  1. 1.Department of Obstetrics and GynaecologyUniversity of Nottingham, City HospitalNottinghamUK
  2. 2.Max-Planck Institute for Physiology and Clinical Research, W.G. Kerckhoff InstituteBad NauheimGermany
  3. 3.Centre for Molecular Biosciences, School of Biomedical SciencesUniversity of UlsterColeraineUK
  4. 4.Department of OphthalmologyQueens Medical CentreNottinghamUK
  5. 5.Department of Medical BiochemistryGothenburg UniversityGothenburgSweden
  6. 6.Institute of Anatomy and Cell Biology, Macroscopic DepartmentJohannes Gutenberg University MainzMainzGermany

Personalised recommendations