Possible Participation of Autocrine and Paracrine Vascular Endothelial Growth Factors in Hypoxia-induced Proliferation of Endothelial Cells and Pericytes

doi:10.1074/jbc.270.47.28316

Possible Participation of Autocrine and Paracrine Vascular Endothelial Growth Factors in Hypoxia-induced Proliferation of Endothelial Cells and Pericytes (*)

From the (1) Departments of Biochemistry and
(2) Neurosurgery, Kanazawa University School of Medicine, 13-1 Takara-machi, Kanazawa 920, Japan

^§ To whom correspondence should be addressed. Tel.: 81-762-62-8151 (ext. 2253); Fax: 81-762-34-4226.

Abstract

Hypoxia is the principal factor that causes angiogenesis. These experiments were conducted to explore how it induces the proliferation of vascular cells, a key step in angiogenesis. Human umbilical vein endothelial cells and bovine retinal pericytes were grown in controlled atmosphere culture chambers containing various concentrations of oxygen. The numbers of both endothelial cells and pericytes increased significantly under hypoxic conditions; the O concentrations that achieved maximal growth promotion were 10% for endothelial cells and 2.5% for pericytes. Quantitative reverse transcription-polymerase chain reaction analysis revealed that mRNAs coding for the secretory forms of vascular endothelial growth factor (VEGF), a mitogen specific to endothelial cells, were present in both endothelial cells and pericytes and that their levels increased significantly in the two cell types as the atmospheric O concentration decreased. The two genes for VEGF receptors, kinase insert domain-containing receptor (kdr) and fms-like tyrosine kinase 1 (flt1), were found to be constitutively expressed in endothelial cells, and their relative mRNA levels were ranked in that order. On the other hand, only flt1 mRNA was detected in pericytes under hypoxic conditions. Furthermore, most antisense oligodeoxyribonucleotides complementary to VEGF mRNAs efficiently inhibited DNA synthesis in endothelial cells cultured under hypoxic conditions. These results indicate that autocrine and paracrine VEGFs may take part in the hypoxia-induced proliferation of endothelial cells.

Footnotes

↵* This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan (to H. Y.), the Japan Diabetes Foundation (to H. Y.), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to H. Y.), and the Hokkoku Foundation for Cancer Research (to H. Y., S. Y., and M. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵⁽⁾ The abbreviations used are:

VEGF

vascular endothelial growth factor

FBS

fetal bovine serum

RT-PCR

reverse transcription-polymerase chain reaction

nt

nucleotide(s)

bp

base pair(s)

bFGF

basic fibroblast growth factor

TGF-β

transforming growth factor-β

PDGF-B

platelet-derived growth factor B.
↵⁽⁾Human umbilical vein endothelial cells responded to a recombinant VEGF added to the medium. Based on a standard curve obtained by plotting radioactivities of incorporated [H]thymidine as a function of the VEGF concentration, 10% O was deduced to be equivalent to 0.15 ng/ml VEGF in the induction of DNA synthesis (M. Nomura and H. Yamamoto, unpublished data). The mitogenic activities of VEGF and endothelial cell growth supplement were not affected by a heparin wash (52) under the present conditions.
- Received October 24, 1994.
- Revision received August 31, 1995.