Am. J. Respir. Cell Mol. Biol., Vol 10, No. 5, May 1994, 552-559.
Characterization of fibroblast mitogens and chemoattractants produced by endothelial cells exposed to hypoxia
KE Dawes, AJ Peacock, AJ Gray, JE Bishop and GJ Laurent
Biochemistry Unit, National Heart and Lung Institute, University of London, United Kingdom.
During pulmonary hypertension there is remodeling of the pulmonary
vasculature, with enhanced fibroblast proliferation and connective tissue
production. The stimulus for this process is not understood, but one
explanation is that endothelial cells secrete moieties that expand local
cell populations by acting as chemoattractants and mitogens. Here, we
investigated the effect of hypoxia (35 mm Hg) on the production of
chemoattractants and mitogens by human umbilical vein endothelial cells.
Endothelial cells were subjected to hypoxia for up to 24 h and the
resultant conditioned media tested for chemotactic and mitogenic activity.
Chemotaxis of pulmonary artery fibroblasts were measured using a 48-well
Boyden chamber and replication assessed by a spectrophotometric method,
based upon the uptake and subsequent elution of methylene blue by
fibroblasts. Within 6 h of culture, media derived from both hypoxic and
normoxic endothelial cells stimulated fibroblast chemotaxis and
replication. This activity increased with time, and by 24 h there was a
significantly greater response toward media obtained from cells exposed to
hypoxia compared with normoxic controls (P < 0.01). The addition of
antibodies to endothelin-1 (Et-1) or platelet- derived growth factor (PDGF)
reduced the chemotactic activity in hypoxic conditioned media by almost 50%
(45 +/- 6 to 24 +/- 5 cells/h.p.f. and 45 +/- 6 to 26 +/- 4.5 cells/h.p.f.
for anti-Et-1 and anti-PDGF, respectively; P < 0.001). Fibroblast
proliferation in response to hypoxic conditioned media was also reduced in
the presence of antibodies to PDGF (55 +/- 11% to 14 +/- 12% above media
control; P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)
