Skip to main content
NCBI home page
The American Journal of Pathology logoLink to The American Journal of Pathology
. 1996 Jan;148(1):129–139.

Direct stimulation of limbal microvessel endothelial cell proliferation and capillary formation in vitro by a corneal-derived eicosanoid.

R A Stoltz 1, M S Conners 1, M E Gerritsen 1, N G Abraham 1, M Laniado-Schwartzman 1
PMCID: PMC1861607  PMID: 8546200

Abstract

12(R)-Hydroxyeicosatrienoic acid (12(R)-HETrE), a corneal epithelial derived inflammatory eicosanoid, elicits blood vessel growth into the avascular cornea in the classical corneal micropocket bioassay. Using an in vivo stimulated angiogenesis assay and 12(R)-HETrE as the angiogenic stimulus, we isolated a homogeneous population of rabbit limbal microvessel endothelial cells, the target for angiogenic factors in the anterior surface of ocular tissues, and analyzed the mitogenic and angiogenic potential of this eicosanoid. 12(R)-HETrE stereospecifically increased cell number by approximately 45%, an effect comparable to that of basic fibroblast growth factor (0.6 nmol/L; 10 ng/ml). This potent mitogenic response was maximal at 0.1 nmol/L. An additive effect (approximately 90% above control) on cell proliferation was observed when 12(R)-HETrE (0.1 nmol/L) and basic fibroblast growth factor (0.6 nmol/L) were added to quiescent cultures of rabbit limbal microvessel endothelial cells. We also show that 12(R)-HETrE, but not 12(S)-HETrE, induces cultured rabbit limbal microvessel endothelial cells to organize themselves as a network of branching cords reminiscent of capillaries. This effect was evident within 48 hours, maximal by 5 days of culture, and paralleled the effect observed with basic fibroblast growth factor. This study describes a novel method for testing site-directed angiogenesis in vitro and further strengthens the angiogenic properties of 12(R)-HETrE by demonstrating a direct effect on limbal microvessel endothelial cells.

Full text

PDF
129

Images in this article

133Figure 1
on p.133
134Figure 2
on p.134
135Figure 4
on p.135
137Figure 7
on p.137

Selected References

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

  1. Alessandri G., Raju K., Gullino P. M. Mobilization of capillary endothelium in vitro induced by effectors of angiogenesis in vivo. Cancer Res. 1983 Apr;43(4):1790–1797. [PubMed] [Google Scholar]
  2. Auerbach R., Alby L., Grieves J., Joseph J., Lindgren C., Morrissey L. W., Sidky Y. A., Tu M., Watt S. L. Monoclonal antibody against angiotensin-converting enzyme: its use as a marker for murine, bovine, and human endothelial cells. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7891–7895. doi: 10.1073/pnas.79.24.7891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carley W. W., Niedbala M. J., Gerritsen M. E. Isolation, cultivation, and partial characterization of microvascular endothelium derived from human lung. Am J Respir Cell Mol Biol. 1992 Dec;7(6):620–630. doi: 10.1165/ajrcmb/7.6.620. [DOI] [PubMed] [Google Scholar]
  4. Chung-Welch N., Patton W. F., Yen-Patton G. P., Hechtman H. B., Shepro D. Phenotypic comparison between mesothelial and microvascular endothelial cell lineages using conventional endothelial cell markers, cytoskeletal protein markers and in vitro assays of angiogenic potential. Differentiation. 1989 Oct;42(1):44–53. doi: 10.1111/j.1432-0436.1989.tb00606.x. [DOI] [PubMed] [Google Scholar]
  5. Conners M. S., Stoltz R. A., Davis K. L., Dunn M. W., Abraham N. G., Levere R. D., Laniado-Schwartzman M. A closed eye contact lens model of corneal inflammation. Part 2: Inhibition of cytochrome P450 arachidonic acid metabolism alleviates inflammatory sequelae. Invest Ophthalmol Vis Sci. 1995 Apr;36(5):841–850. [PubMed] [Google Scholar]
  6. Davis K. L., Conners M. S., Dunn M. W., Schwartzman M. L. Induction of corneal epithelial cytochrome P-450 arachidonate metabolism by contact lens wear. Invest Ophthalmol Vis Sci. 1992 Feb;33(2):291–297. [PubMed] [Google Scholar]
  7. Eliason J. A. Angiogenic activity of the corneal epithelium. Exp Eye Res. 1985 Dec;41(6):721–732. doi: 10.1016/0014-4835(85)90180-0. [DOI] [PubMed] [Google Scholar]
  8. Eliason J. A., Elliott J. P. Proliferation of vascular endothelial cells stimulated in vitro by corneal epithelium. Invest Ophthalmol Vis Sci. 1987 Dec;28(12):1963–1969. [PubMed] [Google Scholar]
  9. Eliason J. A. Leukocytes and experimental corneal vascularization. Invest Ophthalmol Vis Sci. 1978 Nov;17(11):1087–1095. [PubMed] [Google Scholar]
  10. Folkman J. Tumor angiogenesis. Adv Cancer Res. 1985;43:175–203. doi: 10.1016/s0065-230x(08)60946-x. [DOI] [PubMed] [Google Scholar]
  11. Gaffney J., West D., Arnold F., Sattar A., Kumar S. Differences in the uptake of modified low density lipoproteins by tissue cultured endothelial cells. J Cell Sci. 1985 Nov;79:317–325. doi: 10.1242/jcs.79.1.317. [DOI] [PubMed] [Google Scholar]
  12. Gospodarowicz D., Bialecki H., Thakral T. K. The angiogenic activity of the fibroblast and epidermal growth factor. Exp Eye Res. 1979 May;28(5):501–514. doi: 10.1016/0014-4835(79)90038-1. [DOI] [PubMed] [Google Scholar]
  13. Graeber J. E., Glaser B. M., Setty B. N., Jerdan J. A., Walenga R. W., Stuart M. J. 15-Hydroxyeicosatetraenoic acid stimulates migration of human retinal microvessel endothelium in vitro and neovascularization in vivo. Prostaglandins. 1990 Jun;39(6):665–673. doi: 10.1016/0090-6980(90)90026-r. [DOI] [PubMed] [Google Scholar]
  14. Grant D. S., Kleinman H. K., Goldberg I. D., Bhargava M. M., Nickoloff B. J., Kinsella J. L., Polverini P., Rosen E. M. Scatter factor induces blood vessel formation in vivo. Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1937–1941. doi: 10.1073/pnas.90.5.1937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grant D. S., Lelkes P. I., Fukuda K., Kleinman H. K. Intracellular mechanisms involved in basement membrane induced blood vessel differentiation in vitro. In Vitro Cell Dev Biol. 1991 Apr;27A(4):327–336. doi: 10.1007/BF02630910. [DOI] [PubMed] [Google Scholar]
  16. Hoyer L. W., De los Santos R. P., Hoyer J. R. Antihemophilic factor antigen. Localization in endothelial cells by immunofluorescent microscopy. J Clin Invest. 1973 Nov;52(11):2737–2744. doi: 10.1172/JCI107469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jaffe E. A., Hoyer L. W., Nachman R. L. Synthesis of antihemophilic factor antigen by cultured human endothelial cells. J Clin Invest. 1973 Nov;52(11):2757–2764. doi: 10.1172/JCI107471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Johnson A. R., Erdös E. G. Metabolism of vasoactive peptides by human endothelial cells in culture. Angiotensin I converting enzyme (kininase II) and angiotensinase. J Clin Invest. 1977 Apr;59(4):684–695. doi: 10.1172/JCI108687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kinsella J. L., Grant D. S., Weeks B. S., Kleinman H. K. Protein kinase C regulates endothelial cell tube formation on basement membrane matrix, Matrigel. Exp Cell Res. 1992 Mar;199(1):56–62. doi: 10.1016/0014-4827(92)90461-g. [DOI] [PubMed] [Google Scholar]
  20. Laniado-Schwartzman M., Lavrovsky Y., Stoltz R. A., Conners M. S., Falck J. R., Chauhan K., Abraham N. G. Activation of nuclear factor kappa B and oncogene expression by 12(R)-hydroxyeicosatrienoic acid, an angiogenic factor in microvessel endothelial cells. J Biol Chem. 1994 Sep 30;269(39):24321–24327. [PubMed] [Google Scholar]
  21. Madri J. A., Williams S. K. Capillary endothelial cell cultures: phenotypic modulation by matrix components. J Cell Biol. 1983 Jul;97(1):153–165. doi: 10.1083/jcb.97.1.153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Maheshwari R. K., Srikantan V., Bhartiya D., Kleinman H. K., Grant D. S. Differential effects of interferon gamma and alpha on in vitro model of angiogenesis. J Cell Physiol. 1991 Jan;146(1):164–169. doi: 10.1002/jcp.1041460121. [DOI] [PubMed] [Google Scholar]
  23. Marceau F., Boisjoly H. M., Wagner E., Lille S., Roy R. Long-term culture and characterization of human limbal microvascular endothelial cells. Exp Eye Res. 1990 Dec;51(6):645–650. doi: 10.1016/0014-4835(90)90048-y. [DOI] [PubMed] [Google Scholar]
  24. Masferrer J. L., Murphy R. C., Pagano P. J., Dunn M. W., Laniado-Schwartzman M. Ocular effects of a novel cytochrome P-450-dependent arachidonic acid metabolite. Invest Ophthalmol Vis Sci. 1989 Mar;30(3):454–460. [PubMed] [Google Scholar]
  25. Masferrer J. L., Rimarachin J. A., Gerritsen M. E., Falck J. R., Yadagiri P., Dunn M. W., Laniado-Schwartzman M. 12(R)-hydroxyeicosatrienoic acid, a potent chemotactic and angiogenic factor produced by the cornea. Exp Eye Res. 1991 Apr;52(4):417–424. doi: 10.1016/0014-4835(91)90037-f. [DOI] [PubMed] [Google Scholar]
  26. Montesano R., Pepper M. S., Belin D., Vassalli J. D., Orci L. Induction of angiogenesis in vitro by vanadate, an inhibitor of phosphotyrosine phosphatases. J Cell Physiol. 1988 Mar;134(3):460–466. doi: 10.1002/jcp.1041340318. [DOI] [PubMed] [Google Scholar]
  27. Renko M., Quarto N., Morimoto T., Rifkin D. B. Nuclear and cytoplasmic localization of different basic fibroblast growth factor species. J Cell Physiol. 1990 Jul;144(1):108–114. doi: 10.1002/jcp.1041440114. [DOI] [PubMed] [Google Scholar]
  28. Sunderkötter C., Roth J., Sorg C. Immunohistochemical detection of bFGF and TNF-alpha in the course of inflammatory angiogenesis in the mouse cornea. Am J Pathol. 1990 Sep;137(3):511–515. [PMC free article] [PubMed] [Google Scholar]
  29. Voyta J. C., Via D. P., Butterfield C. E., Zetter B. R. Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. J Cell Biol. 1984 Dec;99(6):2034–2040. doi: 10.1083/jcb.99.6.2034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wainwright S., Falck J. R., Yadagiri P., Powell W. S. Metabolism of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid and other hydroxylated fatty acids by the reductase pathway in porcine polymorphonuclear leukocytes. Biochemistry. 1990 Oct 30;29(43):10126–10135. doi: 10.1021/bi00495a017. [DOI] [PubMed] [Google Scholar]
  31. Ziche M., Jones J., Gullino P. M. Role of prostaglandin E1 and copper in angiogenesis. J Natl Cancer Inst. 1982 Aug;69(2):475–482. [PubMed] [Google Scholar]

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

RESOURCES