MM cells as well as PCL serum induce VEC gene and protein expression in monocytes that is blocked by anti-PTN antibodies. (A) Freshly obtained CD14⁺ cells cocultured with MM BM tumor cells with and without anti-PTN antibody were analyzed for Flk-1, Tie-2, and VWF protein expression using Western blot analysis. Haceks served as a positive control. (B) U937, U266, and RPMI8226 cells were analyzed for PTN and GAPDH gene expression with RT-PCR. (C) LAGλ-1 and U266 cells were stained with either anti-PTN or isotype-matched control antibodies. (D) THP-1, U937, RPMI8226, and U266 cells were cultured for 48 hours and the culture supernatant was analyzed for PTN protein concentration by ELISA. Data for PTN graphed are the average of experiments performed in triplicate and show means plus or minus SEM. (E) Endothelial cells (HCAECs) or CD14⁺ cells alone or exposed to M-CSF, the PTN-producing MM cell lines U266 and RPMI8226, or serum from a healthy control subject or a patient with PCL containing high levels of PTN (3.4 ng/mL)²²  were analyzed for gene expression using primers specific for VWF, Tie-2, Flk-1, and GAPDH with RT-PCR. (F) Endothelial cells (HCAECs) or CD14⁺ cells alone or exposed to M-CSF, the PTN-producing MM cell line U266, or BM from a healthy control subject or serum from the patient with PCL were analyzed for expression of the VWF, Tie-2, and Flk-1 genes in the presence of anti-PTN or isotype-matched control antibodies.