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

During pulmonary hypertension there is remodeling of the pulmonary vas culature, 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 loc al 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 cell s. Endothelial cells were subjected to hypoxia for up to 24 h and the resultant conditioned media tested for chemotactic and mitogenic activ ity. Chemotaxis of pulmonary artery fibroblasts was measured using a 4 8-well Boyden chamber and replication assessed by a spectrophotometric method, based upon the uptake and subsequent elution of methylene blu e by fibroblasts. Within 6 h of culture, media derived from both hypox ic 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 exp osed to hypoxia compared with normoxic controls (P < 0.01). The additi on of antibodies to endothelin-1 (Et-1) or platelet-derived growth fac tor (PDGF) reduced the chemotactic activity in hypoxic conditioned med ia 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 < 0.0 01). 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 r adioimmunoassay, in all the media tested, but after exposure to hypoxi a 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, demonstr ated a low molecular weight fraction (3 kD), containing Et-1, which st imulated fibroblast migration, whereas a high molecular weight fractio n (38 kD), containing PDGF, was found to stimulate fibroblast replicat ion and chemotaxis. These results demonstrate that reduced oxygen tens ion stimulates the production of fibroblast mitogens and chemoattracta nts by endothelial cells, and that the bulk of this activity resides i n Et-1 and PDGF. These factors may play an important role in the proli feration and recruitment of fibroblasts seen in pulmonary vascular rem odeling resulting from hypoxia.