American Society of Hematology

Figure 3

$Figure 3. Megakaryocytes drive osteoblast expansion through PDGF-BB expression. (A) BM levels of PDGF-BB, IGF-1, VEGF, SDF-1, and TPO by ELISA (mean ± SD) at baseline and at 48 hours post-TBI in WT mice. *P < .001 vs baseline. (B) Levels of BM PDGF-BB, IGF-1, VEGF, SDF-1, and levels in WT vs mpl−/− mice with or without CD41 or c-MPL antibody blockade (n = 5 per group) at 48 hours post-TBI. *P < .05 vs WT TBI-only group. (C) Representative dot plots showing purity of isolated, ex vivo–expanded megakaryocytes (MEG) and primary osteoblasts (OB) before coculture (left), together with nonadherent (top right) MEG and adherent (bottom right) OB fractions after 5 days of coculture. (D) Five-day growth/survival of MEG and OB cultured separately or in combination (OB+MEG), with or without imatinib (5 μm), anti-CD41 antibody, or anti-c-MPL antibody (n = 3 each; mean ± SD). *P < .01 vs OB cultured alone, +P < .001 vs MEG cultured alone. (E) VEGF, IGF-1, SDF-1, and PDGF-BB supernatant concentrations (mean ± SD, n = 3 each) from 5-day cultures of OB, MEG, or cocultured OB + MEG. *P < .001 vs OB or MEG alone, +P < .001 vs OB alone or OB + MEG. (F) OB growth in 2- and 5-day OB cultures in which either MEG or medium alone was added to cultures across a 0.4-µM transwell membrane (n = 3 each). *P < .002 vs OB cultured with medium alone. (G) Five-day cultures of OB cultured with media alone or with increasing concentrations (2-10 ng/mL) of recombinant PDGF-BB (n = 3 each). *P < .01 vs cultures without PDGF-BB. (H) H&E-stained sections (40×) of BM at 48 hours post-TBI, demonstrating abrogation of OB expansion (arrowheads) caused by imatinib (right) vs sham (left) treatment. (I) Quantitative analysis of average OB layers (left) and percentage of endosteal MEG (right) at 48 hours post-TBI in imatinib vs sham-treated WT mice (n = 5 mice per group). *P < .005 vs sham-treated mice. (J) 40× photomicrographs of H&E-stained metaphyseal BM sections at 48 hours post-TBI in WT and mpl−/− mice demonstrating that treatment with PDGF-BB enhances 48-hour post-TBI endosteal OB proliferation (black arrows) in both WT and mpl−/− BM.$

Megakaryocytes drive osteoblast expansion through PDGF-BB expression. (A) BM levels of PDGF-BB, IGF-1, VEGF, SDF-1, and TPO by ELISA (mean ± SD) at baseline and at 48 hours post-TBI in WT mice. *P < .001 vs baseline. (B) Levels of BM PDGF-BB, IGF-1, VEGF, SDF-1, and levels in WT vs mpl^−/− mice with or without CD41 or c-MPL antibody blockade (n = 5 per group) at 48 hours post-TBI. *P < .05 vs WT TBI-only group. (C) Representative dot plots showing purity of isolated, ex vivo–expanded megakaryocytes (MEG) and primary osteoblasts (OB) before coculture (left), together with nonadherent (top right) MEG and adherent (bottom right) OB fractions after 5 days of coculture. (D) Five-day growth/survival of MEG and OB cultured separately or in combination (OB+MEG), with or without imatinib (5 μm), anti-CD41 antibody, or anti-c-MPL antibody (n = 3 each; mean ± SD). *P < .01 vs OB cultured alone, +P < .001 vs MEG cultured alone. (E) VEGF, IGF-1, SDF-1, and PDGF-BB supernatant concentrations (mean ± SD, n = 3 each) from 5-day cultures of OB, MEG, or cocultured OB + MEG. *P < .001 vs OB or MEG alone, +P < .001 vs OB alone or OB + MEG. (F) OB growth in 2- and 5-day OB cultures in which either MEG or medium alone was added to cultures across a 0.4-µM transwell membrane (n = 3 each). *P < .002 vs OB cultured with medium alone. (G) Five-day cultures of OB cultured with media alone or with increasing concentrations (2-10 ng/mL) of recombinant PDGF-BB (n = 3 each). *P < .01 vs cultures without PDGF-BB. (H) H&E-stained sections (40×) of BM at 48 hours post-TBI, demonstrating abrogation of OB expansion (arrowheads) caused by imatinib (right) vs sham (left) treatment. (I) Quantitative analysis of average OB layers (left) and percentage of endosteal MEG (right) at 48 hours post-TBI in imatinib vs sham-treated WT mice (n = 5 mice per group). *P < .005 vs sham-treated mice. (J) 40× photomicrographs of H&E-stained metaphyseal BM sections at 48 hours post-TBI in WT and mpl^−/− mice demonstrating that treatment with PDGF-BB enhances 48-hour post-TBI endosteal OB proliferation (black arrows) in both WT and mpl^−/− BM.

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