CHARACTERIZATION OF FIBROBLAST MITOGENS AND CHEMOATTRACTANTS PRODUCED BY ENDOTHELIAL-CELLS EXPOSED TO HYPOXIA

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 was 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). Et-1 was detectable, by radioimmunoassay, in all the media tested, but after exposure to hypoxia for 6 or 15 h, conditioned media contained a higher concentration of Et-1 than did normoxic controls (P < 0.05). Separation of endothelial cell conditioned media, using gel filtration chromatography, demonstrated a low molecular weight fraction (3 kD), containing Et-1, which stimulated fibroblast migration, whereas a high molecular weight fraction (38 kD), containing PDGF, was found to stimulate fibroblast replication and chemotaxis. These results demonstrate that reduced oxygen tension stimulates the production of fibroblast mitogens and chemoattractants by endothelial cells, and that the bulk of this activity resides in Et-1 and PDGF. These factors may play an important role in the proliferation and recruitment of fibroblasts seen in pulmonary vascular remodeling resulting from hypoxia.