Abstract

Bone marrow angiogenesis is a hallmark of multiple myeloma (MM). We have recently shown that MM patients express pleitrophin (PTN), a secreted protein that binds syndecan, and it is found at high levels in MM serum. This protein has been shown to stimulate angiogenesis. We have discovered a novel mechanism leading to blood vessel formation by tumor cells. First, we cloned human monocytic THP-1 cells with PTN sense or antisense whole sequencing DNA. We examined expression by RT-PCR of endothelial cell markers (Flk-1), Tie-2, von Willebrand factor (vWf)) and monocyte markers (c-fms and CD68). THP-1 cells infected with PTN sense strand expressed high amounts of Flk-1, Tie-2 and vWf similar to that found in human coronary artery endothelial cells and lost expression of c-fms and CD68. Endothelial cell marker RNA was not detected in either THP-1 cells infected with PTN anti-sense strand or the GFP control vector but these cells showed the continued presence of monocyte markers. Immunohistochemical studies showed THP-1 cells infected with PTN sense strand expressed endothelial markers but not cells treated with antisense or control cells. We have recently found high levels of PTN in MM serum and expression of PTN by MM cell lines including RPMI8226. Next, we cultured THP1 monocytes with RPMI8226 in transwell cultures, serum derived from MM patients with high serum levels of PTN, cell lines lacking PTN expression, and normal controls lacking serum PTN. The THP-1 cells exposed to the MM cell lines or MM serum showed expression of endothelial markers and lost expression of monocyte markers. The expression of endothelial markers was blocked by adding anti-PTN antibody. In contrast, control serum and cell lines lacking PTN expression did not change monocyte marker expression on the THP1 cells nor induce expression of endothelial markers. We examined whether PTN induced monocytes from human peripheral blood to transdifferentiate. Normal human blood monocytes were highly purified using density centrifugation followed by anti-CD14 micro-bead affinity column selection. These purified monocytes also showed transdifferentiation into endothelial cells in the presence of PTN with m-CSF unlike cells treated with m-CSF-alone or cells without these factors present. We determined whether PTN could also stimulate differentiation of bone marrow stem cells into endothelial cells. The stem cells were derived from bone marrow selected for CD34 using magnetic bead selection, and were stimulated with either m-CSF or PTN alone or a combination of m-CSF and PTN or no treatment for 7 days. Real time PCR analysis showed that the m-CSF and PTN combination markedly increased endothelial cell marker expression and decreased monocyte marker (CD68 and c-fms) expression in this stem cell population. When induced with PTN alone, the stem cells exhibited slightly increasing expression of endothelial markers with no change in monocyte marker expression whereas m-CSF alone and no treatment had no effect on either endothelial or monocyte marker expression. These experiments define a previously unrecognized novel mechanism leading to angiogenesis in cancer patients- the transdifferentiation of monocytes into endothelial cells by a factor highly produced by tumor cells. They also suggest a potential new specific target to inhibit angiogenesis-pleitrophin which may have profound clinical implications.

Author notes

Corresponding author