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Hypoxic Induction of Endothelial Cell Growth Factors in Retinal Cells: Identification and Characterization of Vascular Endothelial Growth Factor (VEGF) as the Mitogen

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Abstract

Background

New vessel growth is often associated with ischemia, and hypoxic tissue has been identified as a potential source of angiogenic factors. In particular, ischemia is associated with the development of neovascularization in a number of ocular pathologies. For this reason, we have studied the induction of endothelial cell mitogens by hypoxia in retinal cells.

Materials and Methods

Human retinal pigment epithelium (hRPE) were grown under normoxic and hypoxic conditions and examined for the production of endothelial mitogens. Northern analysis, biosynthetic labeling and immunoprecipitation, and ELISA were used to assess the levels of vascular endothelial growth factor/vascular permeability factor (VEGF) and basic fibroblast growth factor (bFGF), two endothelial cell mitogens and potent angiogenic factors. Soluble receptors for VEGF were employed as competitive inhibitors to determine the contribution of the growth factor to the hypoxia-stimulated mitogen production.

Results

Following 6–24 hr of hypoxia, confluent and growing cultures of hRPE increase their levels of VEGF mRNA and protein synthesis. Biosynthetic labeling studies and RT-PCR analysis indicate that the cells secrete VEGF121 and VEGF165, the soluble forms of the angiogenic factor. In contrast, hRPE cultured under hypoxic conditions show reduced steady-state levels of basic fibroblast growth factor (bFGF) mRNA and decreased bFGF protein synthesis. Unlike VEGF, bFGF is not found in conditioned media of hRPE following 24 hr of hypoxia. Using a soluble high-affinity VEGF receptor as a competitive inhibitor of VEGF, we demonstrate that a VEGF-like activity is the sole hypoxia-inducible endothelial mitogen produced by cultured hRPE.

Conclusions

From this comparison we conclude that hRPE do not respond to hypoxia with a general, nonspecific increase in the overall levels of growth factors, as is seen during cell wounding responses or serum stimulation. The physiological relevance of data from this in vitro model are affirmed by separate studies in an animal model of retinal ischemia-induced ocular neovascularization (1) in which retina-derived VEGF levels have been shown to correlate spatio-temporally with the onset of angiogenesis. Taken together, these data support the hypothesis that the induction of VEGF by hypoxia mediates the rapid, initial angiogenic response to retinal ischemia.

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Acknowledgements

This work was supported by funds from EY05985 (PAD), CA45548 (PAD), EY00325 (APA), and Beth Israel Hospital Pathology Foundation, Inc. (KTY, TKY).

We thank Donald Senger for the anti-VEGF antisera used in the immunoassay, Ying Zhou for the excellent technical assistance, Dr. Harold Dvorak for his helpful comments, Dr. Stella Kourembanas for generously sharing her insights regarding hypoxic regulation of bFGF gene expression, and Sandra R. Smith for her assistance in performing the proliferation assays.

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Authors and Affiliations

  1. Program in Cell and Developmental Biology, Harvard Medical School, Boston, Massachusetts, USA

    David T. Shima & Patricia A. D’Amore

  2. Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA

    Anthony P. Adamis

  3. Department of Surgery, Laboratory for Surgical Research, Children’s Hospital, 300 Longwood Avenue, Boston, MA, 02115, USA

    Anthony P. Adamis, Rapheal Allende, Judah Folkman & Patricia A. D’Amore

  4. Department of Cardiovascular Research, Genentech, Inc., S. San Francisco, California, USA

    Napoleone Ferrara

  5. Department of Pathology, Beth Israel Hospital, Boston, Massachusetts, USA

    Kiang-Teck Yeo & Tet-Kin Yeo

  6. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA

    Judah Folkman

  7. Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA

    Patricia A. D’Amore

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  1. David T. Shima
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Shima, D.T., Adamis, A.P., Ferrara, N. et al. Hypoxic Induction of Endothelial Cell Growth Factors in Retinal Cells: Identification and Characterization of Vascular Endothelial Growth Factor (VEGF) as the Mitogen. Mol Med 1, 182–193 (1995). https://doi.org/10.1007/BF03401566

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