Transdifferentiation of Monocytes into Endothelial Cells by Pleiotrophin.
Authors: Chen Haiming , Campbell Richard A. , Wang Cathy , Li Mingjie , Lee Hee Jin , Yeh Howard S. , Gui Dorina , Gordon Melinda S. , Said Jonathan , Bonavida Benjamin , Berenson James R. 
Published: April 2020

Abstract
Pleiotrophin (PTN) is a secreted angiogenic protein. We have recently shown that malignant plasma cells express high amounts of PTN and that it is elevated in MM patient serum. In this study, we show that this protein leads to angiogenesis through a novel mechanism - transdifferentiation of monocytes into endothelial cells. First, we isolated human monocytes (CD14+ cells) from peripheral blood using immunomagnetic bead isolation. We excluded the presence of endothelial cells in these CD14-expressing cells using RT-PCR on 106 monocytes with primers to genes expressed by endothelial cells including Flk-1, Tie2, CD144, and von Willebrand factor (vWF). We cultured these purified monocytes on collagen I for one week in the presence of PTN, mCSF and VEGF. Cells cultured with the combination of mCSF and PTN developed Flk-1-expressing tube-like structures, and the addition of VEGF increased tube formation. Next, we performed RT-PCR analysis with primers to these endothelial genes on monocytes cultured with PTN, mCSF and VEGF following serial dilution in cells (T or B lymphocytes) that lack monocyte or endothelial cell gene expression. Many of the cells expressed Tie2 RNA (&gt;10%), and a smaller proportion (0.1–1%) also expressed Flk-1, CD144 and vWF RNA. In contrast, purified monocytes incubated with mCSF, VEGF, or PTN alone or the combinations of mCSF and VEGF or PTN and VEGF lacked Flk-1 staining, did not form tubes and failed to express endothelial cell RNA. We also assessed these monocytes in three dimensional matrices using Matrigel. Cells treated with mCSF and PTN invaded the matrix and began to form tube-like structures in the three dimensional gels as early as 7 days following culture whereas monocytes treated with mCSF, VEGF, or PTN alone did not form these structures. Next, we transduced human monocytic THP-1 cells that lack PTN expression with PTN-sense or -anti-sense constructs. Using RT-PCR, THP-1 cells transduced with PTN expressed endothelial cell genes and lost expression of the monocyte genes c-fms and CD68. In contrast, endothelial cell RNA was not detected in either THP-1 cells infected with anti-sense or the GFP control vectors. We used Transwell plates to co-culture THP-1 monocytes with human MM RPMI8226 or U266 cells or cell lines lacking PTN expression. We also added serum from MM patients with high levels of PTN or normal controls lacking PTN to THP-1 cells. THP-1 cells cultured with the MM cell lines or MM serum expressed endothelial genes and lost expression of monocyte RNA. Endothelial gene expression was blocked by the addition of an anti-PTN antibody but not by a control antibody. Control serum and cell lines lacking PTN did not induce endothelial gene expression or changes in monocyte RNA expression in the THP-1 cells. 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 the malignant cells in MM. These findings also suggest a potential new specific target, PTN, to inhibit angiogenesis in cancer patients and should have profound clinical implications.