Circulating Tie2-Expressing Cells Are Increased in Multiple Myeloma Patients, Correlate with Serum Pleiotrophin Levels and May Develop from This Myeloma Angiogenic and Growth Factor.

Abstract

Tie2, an endothelial cell-specific receptor kinase, plays an important role in tumor angiogenesis. This protein is essential to the development of embryonic vasculature as well as vascular growth and maintenance in adult tissues. Because of the increasing importance that angiogenesis has been shown to play in multiple myeloma (MM), we determined the number of Tie2-expressing cells in the peripheral blood (PB) of MM patients and its relationship to the serum levels and gene expression of a recently identified angiogenic factor, pleiotrophin (PTN). We have recently demonstrated that PTN is expressed and secreted by MM tumor cells, and serum levels of this protein are highly elevated in MM patients. We quantified the number of Tie2-positive cells in MM patients (n=15) and age-matched control subjects (n=10) using an immunohistochemical technique. Tie2-expressing cells were significantly elevated in the PB mononuclear cells (MCs) from MM patients compared to the normal controls (p<0.05). We also analyzed gene expression for Tie2 in these same samples using RT-PCR. The results showed that Tie2 mRNA was strongly expressed in the PBMCs from MM patients whereas control samples showed no or low expression of this gene. Serum levels of PTN were tested with ELISA, and PTN mRNA concentrations were quantified by RT-PCR in PBMCs from these same patients and control subjects. The results showed that serum levels of PTN correlated with the number of Tie2-expressing PBMCs in MM patients (R2=0.5778). PTN mRNA levels also correlated with Tie2 gene expression in PBMC samples. We further examined whether monocyte colony stimulating factor (mCSF), PTN and vascular endothelial growth factor (VEGF) may be capable of inducing Tie2 expression in highly purified human monocytes that lack Tie2 expression. Normal PB monocytes were purified using density centrifugation followed by anti-CD14 micro-bead affinity column selection. Although none of these three proteins alone or the combinations of either VEGF and mCSF or VEGF and PTN induced Tie2 gene expression in the monocytes following one week of incubation, the combination of PTN (100 nM) and mCSF (20 nM) led to expression of Tie2 in these cells. We quantified the proportion of cells expressing Tie2 in these samples with RT-PCR using serial dilutional analysis with B or T cells that lack Tie2 expression, and showed that approximately 0.1–1.0% of the monocytes expressed this gene following incubation with PTN and mCSF. Moreover, the addition of VEGF (20 ng/ml) to PTN and mCSF increased the proportion of cells expressing Tie2 (to >10%). Anti-PTN antibody blocked the induction of Tie2 gene expression in these monocytes by this cytokine combination. These results show that Tie2-expressing cells are elevated in the peripheral blood of MM patients, and correlate with PTN serum and PTN mRNA expression. PTN in combination with VEGF and mCSF induces Tie2 gene expression in a large proportion of circulating human monocytes. These results suggest that MM patients show increased numbers of vasculogenic progenitors in their circulation that may result from the presence of elevated levels of circulating angiogenic factors including PTN and VEGF.