Suppression of proliferation by endothelial cell-conditioned media is due in part to presence of heparin-binding macromolecule. (a) In further experiments, HMEC-1 were cultured on 96-well plates in the presence of 10% FCS to promote proliferation. Treatment with mitomycin C at baseline was used as a negative control. Addition of conditioned media from hypoxic HMEC-1 blocked proliferation. (b) Filtration to remove molecules > 10 kDa, or heparin binding molecules blocked the effect of hypoxia conditioned media on morphology of treated cells. (c) In further experiments, filtration was used to remove molecules > 100 kDa or (d) immunoprecipitation of TSP-1 antagonised the antiproliferative effects of the conditioned media.