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. 2020 Oct 14;10(1):17319.
doi: 10.1038/s41598-020-74352-x.

Osteocyte Vegf-a contributes to myeloma-associated angiogenesis and is regulated by Fgf23

Patrick L Mulcrone  1   2 , Shanique K E Edwards  3 , Daniela N Petrusca  4 , Laura S Haneline  3 , Jesús Delgado-Calle  4   5   2 , G David Roodman  6   7   8
Affiliations

Osteocyte Vegf-a contributes to myeloma-associated angiogenesis and is regulated by Fgf23

Patrick L Mulcrone et al. Sci Rep. .

Abstract

Multiple Myeloma (MM) induces bone destruction, decreases bone formation, and increases marrow angiogenesis in patients. We reported that osteocytes (Ocys) directly interact with MM cells to increase tumor growth and expression of Ocy-derived factors that promote bone resorption and suppress bone formation. However, the contribution of Ocys to enhanced marrow vascularization in MM is unclear. Since the MM microenvironment is hypoxic, we assessed if hypoxia and/or interactions with MM cells increases pro-angiogenic signaling in Ocys. Hypoxia and/or co-culture with MM cells significantly increased Vegf-a expression in MLOA5-Ocys, and conditioned media (CM) from MLOA5s or MM-MLOA5 co-cultured in hypoxia, significantly increased endothelial tube length compared to normoxic CM. Further, Vegf-a knockdown in MLOA5s or primary Ocys co-cultured with MM cells or neutralizing Vegf-a in MM-Ocy co-culture CM completely blocked the increased endothelial activity. Importantly, Vegf-a-expressing Ocy numbers were significantly increased in MM-injected mouse bones, positively correlating with tumor vessel area. Finally, we demonstrate that direct contact with MM cells increases Ocy Fgf23, which enhanced Vegf-a expression in Ocys. Fgf23 deletion in Ocys blocked these changes. These results suggest hypoxia and MM cells induce a pro-angiogenic phenotype in Ocys via Fgf23 and Vegf-a signaling, which can promote MM-induced marrow vascularization.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Hypoxia Increases Vegf-a Levels in MLOA5s, MLOY4s, and Primary Calvarial Osteocytes. (a) Confirmation of gene expression changes for Vascular Endothelial Growth Factor A (Vegf-a) in MLOA5s. N = 3–4. (b) Increased Vegf-a is secreted into media of hypoxic MLOA5s. N = 6. (c) Representative western blot images of normoxic (Lane 1) and hypoxic (Lanes 2, 3 biological replicates) MLOA5 osteocytes run on the same gel cropped for publication purposes. N = 4 for Western blot analysis. Full-length gel is provided as Supplemental Fig. S8. (d) qPCR for Vegf-a in MLOY4s cultured in Normoxia or Hypoxia (1%) for 24 h. (e) ELISA for Vegf-a from conditioned media of MLOY4s. N = 3–4 for both studies. (f) qPCR for Vegf-a in Primary Calvarial Osteocytes cultured in Normoxia or Hypoxia (1%) for 24 h. (g) ELISA for Vegf-a from conditioned media of Primary Calvarial Osteocytes. N = 4 for both studies. (* = p < 0.05, ** = p < 0.01).
Figure 2
Figure 2
Addition of anti-Vegf-a Antibody to Hypoxic Osteocyte Conditioned Media Reduces Tube Formation in vitro. (a) Representative 4 × images of 5000 HUVECs. Hypoxic conditioned media (CM) group exhibits greater vascular complexity compared to other 3 groups. (b) Branching point analysis and (c) Tube length calculations are increased in Hypoxic CM treated HUVECs. N = 5. (d) and (e) A similar pattern is observed using primary Endothelial Colony Formation Cells (ECFCs). (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001 by ANOVA).
Figure 3
Figure 3
Co-culture of Human MM in Either Normoxia or Hypoxia Increases Vegf-a Secretion by MLOA5 Osteocytes after 24 h. (a) Murine Vegf-a levels detected by ELISA from varying conditioned media. MM cell lines used: RPMIs, MM1.S, JJN3s. N = 4–6. (b) A similar pattern is seen with 5TGM1 murine MM co-cultures. N = 3–4, (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).
Figure 4
Figure 4
Mice Harboring MM Have Increased Percentage of Vegf-a-positive Osteocytes, which Correlates with Tumor Vessel Area. (a) Representative 40 × images, from two separate mice in each group, of Vegf-a-stained murine tibiae injected intratibially with saline or 105 5TGM1 multiple myeloma (MM) cells. Dotted black box indicates zoomed in area of each section. Black arrows indicate Vegf-a-positive osteocytes. (b) Percentage of Vegfa-positive osteocytes 4 weeks after inoculation. N = 8 for each group. (*** = p < 0.001). (c) Representative 20 × images of tibiae from two separate mice in each group, stained with Endomucin. Dotted black box indicates zoomed in area of each section. Black arrows indicate Endomucin-positive blood vessels. (d) Correlation between percent Vegf-a-positive osteocytes and Endomucin-positive tumor vessel area in 5TGM1 MM-bearing mice. (N = 6, p = 0.058).
Figure 5
Figure 5
Knockdown of Vegf-a in Hypoxic Osteocytes Blunts HUVEC Vessel Formation in vitro. (a) Representative 4 × images with zoomed in insert of HUVEC networks. (b) Branching point and C) Tube Length analyses reveal increased vascular complexity in Scr-Hypoxia group compared to other 3 groups. N = 3 (**** = p < 0.0001 by ANOVA).
Figure 6
Figure 6
Conditioned Media from MM:siVegfa-MLOA5 Co-Cultures Does Not Induce HUVEC Vessel Formation: (a) Fold Change in branching point analysis of HUVECs treated with RPMI:MLOA5 co-culture CM at 8 h. N = 4. (b) Fold Change in branching point analysis of HUVECs treated with 5TGM1:MLOA5 co-culture CM at 8 h. N = 3, (* = p < 0.05, ** = p < 0.01, *** = p < 0.001 by ANOVA).
Figure 7
Figure 7
Knockdown of Osteocytic Vegf-a Reduces Tube Formation and MM Expression of VEGF-A and CCND1 in EVOCA:MM Co-Culture Model: (a) Representative 10 × images of HUVECs treated with hypoxic CM from designated co-culture groups. (b) Quantification of branching points. N = 3. (c) Murine Vegf-a expression shows multiplicity of infection (MOI) of 120 reduces murine Vegf-a in primary osteocytes N = 3. (d) Expression of human VEGF-A and CCND1 do not increase in RPMIs and JJN3s co-cultured with Cre-treated bone nor in MM mono-culture controls. Murine Vegf-a levels are decreased with Cre transduction. N = 3–6. (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001).
Figure 8
Figure 8
Fgf23 Enhances Vegf-a Expression and Secretion by Osteocytes. (a) RPMI (Gray), JJN3 (Blue), & MM1.S (Green) myeloma cells induce Fgf23 expression in MLOA5s. N = 3–6. (b) Egr1 expression in MLOA5s increases with 100 ng/mL of Fgf23 treatment and is reduced with 50 nM BGJ398 treatment. (c) Vegf-a expression in MLOA5s follows the same pattern. N = 3–4 for B & C. Vehicle treatment time is 24 h. (d) Vegf-a ELISA shows Fgf23 Induces Secretion of Vegf-a in MLOA5 Osteocytes. This is reduced with 50 nM BGJ398 at 6 h. N = 6. (e) Osteocyte-enriched calvaria from Fgf23cKO mice secrete less Vegf-a in the presence of JJN3s via ELISA. N = 9. (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).
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