Skip to main content
PMC home page
The American Journal of Pathology logoLink to The American Journal of Pathology
. 1998 Jun;152(6):1667–1679.

Mouse model of angiogenesis.

T Couffinhal 1, M Silver 1, L P Zheng 1, M Kearney 1, B Witzenbichler 1, J M Isner 1
PMCID: PMC1858441  PMID: 9626071

Abstract

Neovascularization of ischemic muscle may be sufficient to preserve tissue integrity and/or function and may thus be considered to be therapeutic. The regulatory role of vascular endothelial growth factor (VEGF) in therapeutic angiogenesis was suggested by experiments in which exogenously administered VEGF was shown to augment collateral blood flow in animals and patients with experimentally induced hindlimb or myocardial ischemia. To address the possible contribution of postnatal endogenous VEGF expression to collateral vessel development in ischemia tissues, we developed a mouse model of hindlimb ischemia. The femoral artery of one hindlimb was ligated and excised. Laser Doppler perfusion imaging (LDPI) was employed to document the consequent reduction in hindlimb blood flow, which typically persisted for up to 7 days. Serial in vivo examinations by LDPI disclosed that hindlimb blood flow was progressively augmented over the course of 14 days, ultimately reaching a plateau between 21 and 28 days. Morphometric analysis of capillary density performed at the same time points selected for in vivo analysis of blood flow by LDPI confirmed that the histological sequence of neovascularization corresponded temporally to blood flow recovery detected in vivo. Endothelial cell proliferation was documented by immunostaining for bromodeoxyuridine injected 24 hours before each of these time points, providing additional evidence that angiogenesis constitutes the basis for improved collateral-dependent flow in this animal model. Neovascularization was shown to develop in association with augmented expression of VEGF mRNA and protein from skeletal myocytes as well as endothelial cells in the ischemic hindlimb; that such reparative angiogenesis is indeed dependent upon VEGF up-regulation was confirmed by impaired neovascularization after administration of a neutralizing VEGF antibody. Sequential characterization of the in vivo, histological, and molecular findings in this novel animal model thus document the role of VEGF as endogenous regulator of angiogenesis in the setting of tissue ischemia. Moreover, this murine model represents a potential means for studying the effects of gene targeting on nutrient angiogenesis in vivo.

Full text

PDF
1667

Images in this article

1671Figure 1
on p.1671
1673Figure 3
on p.1673
1674Figure 5
on p.1674
1675Figure 6
on p.1675
1676Figure 7
on p.1676
1677Figure 8
on p.1677
1678Figure 9
on p.1678

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adamis A. P., Miller J. W., Bernal M. T., D'Amico D. J., Folkman J., Yeo T. K., Yeo K. T. Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy. Am J Ophthalmol. 1994 Oct 15;118(4):445–450. doi: 10.1016/s0002-9394(14)75794-0. [DOI] [PubMed] [Google Scholar]
  2. Adamis A. P., Shima D. T., Tolentino M. J., Gragoudas E. S., Ferrara N., Folkman J., D'Amore P. A., Miller J. W. Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization in a nonhuman primate. Arch Ophthalmol. 1996 Jan;114(1):66–71. doi: 10.1001/archopht.1996.01100130062010. [DOI] [PubMed] [Google Scholar]
  3. Aiello L. P., Avery R. L., Arrigg P. G., Keyt B. A., Jampel H. D., Shah S. T., Pasquale L. R., Thieme H., Iwamoto M. A., Park J. E. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med. 1994 Dec 1;331(22):1480–1487. doi: 10.1056/NEJM199412013312203. [DOI] [PubMed] [Google Scholar]
  4. Banai S., Jaklitsch M. T., Shou M., Lazarous D. F., Scheinowitz M., Biro S., Epstein S. E., Unger E. F. Angiogenic-induced enhancement of collateral blood flow to ischemic myocardium by vascular endothelial growth factor in dogs. Circulation. 1994 May;89(5):2183–2189. doi: 10.1161/01.cir.89.5.2183. [DOI] [PubMed] [Google Scholar]
  5. Baumgartner I., Pieczek A., Manor O., Blair R., Kearney M., Walsh K., Isner J. M. Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia. Circulation. 1998 Mar 31;97(12):1114–1123. doi: 10.1161/01.cir.97.12.1114. [DOI] [PubMed] [Google Scholar]
  6. Bauters C., Asahara T., Zheng L. P., Takeshita S., Bunting S., Ferrara N., Symes J. F., Isner J. M. Physiological assessment of augmented vascularity induced by VEGF in ischemic rabbit hindlimb. Am J Physiol. 1994 Oct;267(4 Pt 2):H1263–H1271. doi: 10.1152/ajpheart.1994.267.4.H1263. [DOI] [PubMed] [Google Scholar]
  7. Breen E. C., Johnson E. C., Wagner H., Tseng H. M., Sung L. A., Wagner P. D. Angiogenic growth factor mRNA responses in muscle to a single bout of exercise. J Appl Physiol (1985) 1996 Jul;81(1):355–361. doi: 10.1152/jappl.1996.81.1.355. [DOI] [PubMed] [Google Scholar]
  8. Carmeliet P., Ferreira V., Breier G., Pollefeyt S., Kieckens L., Gertsenstein M., Fahrig M., Vandenhoeck A., Harpal K., Eberhardt C. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature. 1996 Apr 4;380(6573):435–439. doi: 10.1038/380435a0. [DOI] [PubMed] [Google Scholar]
  9. Couffinhal T., Kearney M., Witzenbichler B., Chen D., Murohara T., Losordo D. W., Symes J., Isner J. M. Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in normal and atherosclerotic human arteries. Am J Pathol. 1997 May;150(5):1673–1685. [PMC free article] [PubMed] [Google Scholar]
  10. Duplàa C., Couffinhal T., Dufourcq P., Llanas B., Moreau C., Bonnet J. The integrin very late antigen-4 is expressed in human smooth muscle cell. Involvement of alpha 4 and vascular cell adhesion molecule-1 during smooth muscle cell differentiation. Circ Res. 1997 Feb;80(2):159–169. doi: 10.1161/01.res.80.2.159. [DOI] [PubMed] [Google Scholar]
  11. Ferrara N., Carver-Moore K., Chen H., Dowd M., Lu L., O'Shea K. S., Powell-Braxton L., Hillan K. J., Moore M. W. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature. 1996 Apr 4;380(6573):439–442. doi: 10.1038/380439a0. [DOI] [PubMed] [Google Scholar]
  12. Folkman J., Klagsbrun M. Angiogenic factors. Science. 1987 Jan 23;235(4787):442–447. doi: 10.1126/science.2432664. [DOI] [PubMed] [Google Scholar]
  13. Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis. N Engl J Med. 1995 Dec 28;333(26):1757–1763. doi: 10.1056/NEJM199512283332608. [DOI] [PubMed] [Google Scholar]
  14. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med. 1971 Nov 18;285(21):1182–1186. doi: 10.1056/NEJM197111182852108. [DOI] [PubMed] [Google Scholar]
  15. Frank S., Hübner G., Breier G., Longaker M. T., Greenhalgh D. G., Werner S. Regulation of vascular endothelial growth factor expression in cultured keratinocytes. Implications for normal and impaired wound healing. J Biol Chem. 1995 May 26;270(21):12607–12613. doi: 10.1074/jbc.270.21.12607. [DOI] [PubMed] [Google Scholar]
  16. Freeman M. R., Schneck F. X., Gagnon M. L., Corless C., Soker S., Niknejad K., Peoples G. E., Klagsbrun M. Peripheral blood T lymphocytes and lymphocytes infiltrating human cancers express vascular endothelial growth factor: a potential role for T cells in angiogenesis. Cancer Res. 1995 Sep 15;55(18):4140–4145. [PubMed] [Google Scholar]
  17. Hang J., Kong L., Gu J. W., Adair T. H. VEGF gene expression is upregulated in electrically stimulated rat skeletal muscle. Am J Physiol. 1995 Nov;269(5 Pt 2):H1827–H1831. doi: 10.1152/ajpheart.1995.269.5.H1827. [DOI] [PubMed] [Google Scholar]
  18. Hariawala M. D., Horowitz J. R., Esakof D., Sheriff D. D., Walter D. H., Keyt B., Isner J. M., Symes J. F. VEGF improves myocardial blood flow but produces EDRF-mediated hypotension in porcine hearts. J Surg Res. 1996 Jun;63(1):77–82. doi: 10.1006/jsre.1996.0226. [DOI] [PubMed] [Google Scholar]
  19. Ikeda E., Achen M. G., Breier G., Risau W. Hypoxia-induced transcriptional activation and increased mRNA stability of vascular endothelial growth factor in C6 glioma cells. J Biol Chem. 1995 Aug 25;270(34):19761–19766. doi: 10.1074/jbc.270.34.19761. [DOI] [PubMed] [Google Scholar]
  20. Isner J. M., Pieczek A., Schainfeld R., Blair R., Haley L., Asahara T., Rosenfield K., Razvi S., Walsh K., Symes J. F. Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischaemic limb. Lancet. 1996 Aug 10;348(9024):370–374. doi: 10.1016/s0140-6736(96)03361-2. [DOI] [PubMed] [Google Scholar]
  21. Levy A. P., Levy N. S., Wegner S., Goldberg M. A. Transcriptional regulation of the rat vascular endothelial growth factor gene by hypoxia. J Biol Chem. 1995 Jun 2;270(22):13333–13340. doi: 10.1074/jbc.270.22.13333. [DOI] [PubMed] [Google Scholar]
  22. Lindén M., Sirsjö A., Lindbom L., Nilsson G., Gidlöf A. Laser-Doppler perfusion imaging of microvascular blood flow in rabbit tenuissimus muscle. Am J Physiol. 1995 Oct;269(4 Pt 2):H1496–H1500. doi: 10.1152/ajpheart.1995.269.4.H1496. [DOI] [PubMed] [Google Scholar]
  23. Maione T. E., Gray G. S., Petro J., Hunt A. J., Donner A. L., Bauer S. I., Carson H. F., Sharpe R. J. Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides. Science. 1990 Jan 5;247(4938):77–79. doi: 10.1126/science.1688470. [DOI] [PubMed] [Google Scholar]
  24. Mukhopadhyay D., Tsiokas L., Zhou X. M., Foster D., Brugge J. S., Sukhatme V. P. Hypoxic induction of human vascular endothelial growth factor expression through c-Src activation. Nature. 1995 Jun 15;375(6532):577–581. doi: 10.1038/375577a0. [DOI] [PubMed] [Google Scholar]
  25. Namiki A., Brogi E., Kearney M., Kim E. A., Wu T., Couffinhal T., Varticovski L., Isner J. M. Hypoxia induces vascular endothelial growth factor in cultured human endothelial cells. J Biol Chem. 1995 Dec 29;270(52):31189–31195. doi: 10.1074/jbc.270.52.31189. [DOI] [PubMed] [Google Scholar]
  26. Nicosia R. F., Lin Y. J., Hazelton D., Qian X. Endogenous regulation of angiogenesis in the rat aorta model. Role of vascular endothelial growth factor. Am J Pathol. 1997 Nov;151(5):1379–1386. [PMC free article] [PubMed] [Google Scholar]
  27. Pearlman J. D., Hibberd M. G., Chuang M. L., Harada K., Lopez J. J., Gladstone S. R., Friedman M., Sellke F. W., Simons M. Magnetic resonance mapping demonstrates benefits of VEGF-induced myocardial angiogenesis. Nat Med. 1995 Oct;1(10):1085–1089. doi: 10.1038/nm1095-1085. [DOI] [PubMed] [Google Scholar]
  28. Plate K. H., Breier G., Weich H. A., Risau W. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature. 1992 Oct 29;359(6398):845–848. doi: 10.1038/359845a0. [DOI] [PubMed] [Google Scholar]
  29. Sharpe R. J., Byers H. R., Scott C. F., Bauer S. I., Maione T. E. Growth inhibition of murine melanoma and human colon carcinoma by recombinant human platelet factor 4. J Natl Cancer Inst. 1990 May 16;82(10):848–853. doi: 10.1093/jnci/82.10.848. [DOI] [PubMed] [Google Scholar]
  30. Takeshita S., Tsurumi Y., Couffinahl T., Asahara T., Bauters C., Symes J., Ferrara N., Isner J. M. Gene transfer of naked DNA encoding for three isoforms of vascular endothelial growth factor stimulates collateral development in vivo. Lab Invest. 1996 Oct;75(4):487–501. [PubMed] [Google Scholar]
  31. Takeshita S., Zheng L. P., Brogi E., Kearney M., Pu L. Q., Bunting S., Ferrara N., Symes J. F., Isner J. M. Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. J Clin Invest. 1994 Feb;93(2):662–670. doi: 10.1172/JCI117018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Taussig R., Gilman A. G. Mammalian membrane-bound adenylyl cyclases. J Biol Chem. 1995 Jan 6;270(1):1–4. doi: 10.1074/jbc.270.1.1. [DOI] [PubMed] [Google Scholar]
  33. Tsurumi Y., Murohara T., Krasinski K., Chen D., Witzenbichler B., Kearney M., Couffinhal T., Isner J. M. Reciprocal relation between VEGF and NO in the regulation of endothelial integrity. Nat Med. 1997 Aug;3(8):879–886. doi: 10.1038/nm0897-879. [DOI] [PubMed] [Google Scholar]
  34. Tsurumi Y., Takeshita S., Chen D., Kearney M., Rossow S. T., Passeri J., Horowitz J. R., Symes J. F., Isner J. M. Direct intramuscular gene transfer of naked DNA encoding vascular endothelial growth factor augments collateral development and tissue perfusion. Circulation. 1996 Dec 15;94(12):3281–3290. doi: 10.1161/01.cir.94.12.3281. [DOI] [PubMed] [Google Scholar]
  35. Unthank J. L., Nixon J. C., Lash J. M. Early adaptations in collateral and microvascular resistances after ligation of the rat femoral artery. J Appl Physiol (1985) 1995 Jul;79(1):73–82. doi: 10.1152/jappl.1995.79.1.73. [DOI] [PubMed] [Google Scholar]
  36. Wårdell K., Jakobsson A., Nilsson G. E. Laser Doppler perfusion imaging by dynamic light scattering. IEEE Trans Biomed Eng. 1993 Apr;40(4):309–316. doi: 10.1109/10.222322. [DOI] [PubMed] [Google Scholar]
  37. Yuan F., Chen Y., Dellian M., Safabakhsh N., Ferrara N., Jain R. K. Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody. Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14765–14770. doi: 10.1073/pnas.93.25.14765. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The American Journal of Pathology are provided here courtesy of American Society for Investigative Pathology

RESOURCES