Angiopoietin-1 and Osteopontin Expression by CD138+ Myeloma Cells Rather Than VEGF and CD45 Correlates with Bone Marrow Angiogenesis in Multiple Myeloma Patients at the Diagnosis.
Authors: Giuliani Nicola , Colla Simona , Morandi Francesca , Bonomini Sabrina , Lazzaretti Mirca , Mancini Cristina , Crugnola Monica , Tagliaferri Sara , Rizzato Rita , Martella Eugenia , Sammarelli Gabriella , Craviotto Luisa , Caramatti Cecilia , Mangoni Marcellina , Rizzoli Vittorio 
Published: April 2020

Abstract
Bone marrow (BM) angiogenesis is increased in Multiple Myeloma (MM) patients and correlates with disease progression and patient survival. Myeloma cells secrete the main endothelial growth factor VEGF. In mouse models VEGF secretion as well as the angiogenic properties of MM cells correlate with the lack of CD45 expression by MM cells. However, recent data indicate that VEGF plasma cell expression is similar between MGUS and MM patients suggesting that other molecules could be involved. In line with this hypothesis we have recently demonstrated that myeloma cells may also produce factors with angiogenic properties as angiopoietin-1 (ANG-1) and osteopontin (OPN) that are involved in myeloma induced angiogenesis in vitro.
In order to identify which factors correlate with BM angiogenesis in MM patients, we have investigated in a cohort of 121 newly diagnosed MM patients (stage I–III) the expression of the angiogenic molecules VEGF, ANG-1 and OPN and their correlation with bone marrow (BM) angiogenesis and CD45 expression by MM cells.
We found that 90% of CD138+ MM cells tested were positive for VEGF mRNA. On the other hand we found that 50% and 40 % of MM patients were positive for ANG-1 and OPN mRNA respectively. Using the previously published cut off for CD45 expression we found that 61 out of 121 MM patients were positive for CD45 and 60 out of 121 were negative for CD45 expression.
Any correlation was not observed between VEGF expression and BM angiogenesis in MM patients (p=0.5), whereas the number of microvessels X field was higher in Ang-1 positive patients in comparison with Ang-1 negative ones (mean±SE: 6.23±0.2 vs. 2.94±0.1, median: 6.21 vs. 2.79; p=0.001,) and the microvascular density (MVD) was significantly increased (32.98±1.7 vs. 14.55±1.3, median: 34.69 vs. 13.04; p&lt;0.01; capillaries: 26.73±1.3 vs. 10.42±0.8, median: 24.06 vs. 9.04; p&lt;0.01, small venules: 9.56 ±0.5 vs. 4.14±0.5, median: 10.60 vs. 3.65; p&lt;0.01). Furthermore a significantly positive correlation between Ang-1 expression and MVD was found (Pearson Chi-square: p=0.036, Cochran’s Linear Trend: p=0.01). A significantly higher MVD was also observed in the group of patients positive for OPN, (mean±SE: 29.1±0.7 vs. 17.55±0.37; p&lt;0.01) and similarly, the number of microvessels per field was higher in OPN positive patients in comparison with OPN negative ones (mean±SE: 6.7±0.15 vs. 4.28±0.04; p=0.05). On the other hand, any significant difference was not observed between CD45 positive and CD45 negative patients for the expression of VEGF (p=0.4), ANG-1 (p=0.3) and OPN (p=0.09). Consistently we did not find any significant difference in both MVD and number of vessels X field between CD45 positive patients as compared with CD45 negative ones (p=0.5 and p=0.4, respectively). Finally, a multivariate analysis confirmed that VEGF and CD45 did not correlate with the BM angiogenesis showing that ANG-1 expression by MM cells was more tightly correlated with MVD and the number of vessels X field as compared to OPN.
Our data indicate that ANG-1 and in part OPN rather than VEGF and CD45 expression by MM cells are the critical determinants correlated with the increase of BM angiogenesis that occurs in MM patients at the diagnosis.