Proliferation of DMVECs in response to free heme. The proliferative response of uninfected and KSHV-infected cells to heme was determined by direct cell counts. (A) Cells were plated in complete media overnight followed by culture in serum-free media (SFM) for 24 hours in the presence or absence of the heme analogs CrMP (to inhibit HO-1 activity) and CuMP (noninhibitory). Cells were then incubated with and without 5 μM heme in SFM and proliferation measured 48 hours later. Uninfected cells (□) did not proliferate in response to incubation with heme, whereas KSHV-infected cells (▪) proliferated to a greater extent following exposure to heme. Enhanced proliferation of KSHV-infected cells in the presence of heme was HO-dependent, since treatment with CrMP abolished the proliferative response. (B) The lack of proliferation of uninfected cells was due to an unresponsiveness to heme rather than a nonspecific limitation resulting from the experimental procedure employed. These cells did exhibit a robust proliferative response when serum-starved cells were incubated with 10% human serum in the presence or absence of endothelial cell growth supplement (ECGS; 50 μg/mL), a complex mixture containing known endothelial cell mitogens. Results were determined in quadruplicate (error bars indicate ± SD).