Bortezomib impairs marrow and spleen fibrosis development in TPO^high mice through TGF-β1 inhibition. (A-C) Bortezomib decreases (A) TGF-β1 plasma levels and (B) the total form of TGF-β1 as well as (C) its active form in extracellular fluids of marrow of TPO^high mice in a dose-dependent manner with respective P_(DE) values ≤ .001, .010, and ≤ .001. Total TGF-β1 (active + latent forms) levels were quantified in (A) plasma and in (B) extracellular fluids using an ELISA after acidification of the sample. (C) Active TGF-β1 levels in extracellular fluids were determined without acidification. Note that the media supplemented with 10% fetal calf serum (FCS) used to prepare extracellular fluids of bone marrow contain less than 1.5 ng/mL TGF-β1 and no active form. No spontaneously active TGF-β1 was detected before acidification of the plasma samples. Results in plasma and in extracellular fluids are presented as the mean ± SEM of 12 (except for the “1 mg/kg” group that included only 6 surviving mice at week 8) and of 3 animals per experimental group, respectively (similar results were obtained in 3 other animals per experimental group at week 16 after engraftment, except for the “1 mg/kg” group). Results of statistical analysis with the Wilcoxon test are as follows: treated (bortezomib 0.25 mg/kg, 0.5 mg/kg, and 1 mg/kg) versus untreated (vehicle) mice; *P < .05; **P < .001. Bzb indicates bortezomib. (D-L) Bortezomib impairs marrow and spleen fibrosis development in TPO^high mice after 4 weeks of treatment (8 weeks after engraftment). (D) Representative femora of control mice (left), untreated (vehicle) TPO^high mice (middle), and treated (bortezomib 0.5 mg/kg) TPO^high mice (right). Image was obtained using an Olympus SZX12 stereomicroscope with 1.2× objective (magnification × 8.4), a DP50 Olympus digital camera and the analySIS© acquisition software (Olympus, Tokyo, Japan). Representative histologic sections of femora (E-H) and spleen (I-L) from treated (bortezomib 1 mg/kg; G-H, K-L) or untreated (vehicle; E-F, I-J) TPO^high mice are shown. Bone marrow and spleen sections stained by hematoxylin and eosin show the hyperplasia of megakaryocytes and granulocytes in both treated (G, K) and untreated (E, I) mice. Silver staining reveals (F) marrow and (J) spleen fibrosis in untreated mice with reticulin fibers surrounding the megakaryocytes. Bortezomib treatment impairs reticulin deposition in both bone (H) marrow and (L) spleen. Original magnification × 250.