Activation of the hypoxia-inducible factor-1alpha pathway accelerates bone regeneration

The hypoxia-inducible factor-1alpha (HIF-1alpha) pathway is the central regulator of adaptive responses to low oxygen availability and is required for normal skeletal development. Here, we demonstrate that the HIF-1alpha pathway is activated during bone repair and can be manipulated genetically and pharmacologically to improve skeletal healing. Mice lacking pVHL in osteoblasts with constitutive HIF-1alpha activation in osteoblasts had markedly increased vascularity and produced more bone in response to distraction osteogenesis, whereas mice lacking HIF-1alpha in osteoblasts had impaired angiogenesis and bone healing. The increased vascularity and bone regeneration in the pVHL mutants were VEGF dependent and eliminated by concomitant administration of VEGF receptor antibodies. Small-molecule inhibitors of HIF prolyl hydroxylation stabilized HIF/VEGF production and increased angiogenesis in vitro. One of these molecules (DFO) administered in vivo into the distraction gap increased angiogenesis and markedly improved bone regeneration. These results identify the HIF-1alpha pathway as a critical mediator of neoangiogenesis required for skeletal regeneration and suggest the application of HIF activators as therapies to improve bone healing.     

Lymphoid-restricted development from multipotent candidate murine stem cells: distinct and complimentary functions of the c-kit and flt3-ligands

The two tyrosine kinase receptors, c-kit and flt3, and their respective ligands KL and FL, have been demonstrated to play key and nonredundant roles in regulating the earliest events in hematopoiesis. However, their precise roles and potential interactions in promoting early lymphoid commitment and development remain unclear. Here we show that most if not all murine Lin(-/lo)Sca1(+)c-kit(+) bone marrow (BM) cells generating B220(+)CD19(+) proB-cells in response to FL and interleukin-7 (IL-7) also have a myeloid potential. In contrast to FL + IL-7, KL + IL-7 could not promote proB-cell formation from Lin(-/lo)Sca1(+)c-kit(+) cells. However, KL potently enhanced FL + IL-7-stimulated proB-cell formation, in part through enhanced recruitment of FL + IL-7-unresponsive Lin(-/lo)Sca1(+)c-kit(+) progenitors, and in part by enhancing the growth of proB-cells. The enhanced recruitment (4-fold) in response to KL occurred exclusively from the Lin(-/lo)Sca1(+)c-kit(+)flt3(-) long-term repopulating stem cell population, whereas KL had no effect on FL + IL-7-stimulated recruitment of Lin(-/lo)Sca1(+)c-kit(+)flt3(+) short-term repopulating cells. The progeny of FL + IL-7-stimulated Lin(-/lo)Sca1(+)c-kit(+) cells lacked in vitro and in vivo myeloid potential, but efficiently reconstituted both B and T lymphopoiesis. In agreement with this FL, but not KL, efficiently induced expression of B220 and IL-7 receptor-alpha on Lin(-/lo)Sca1(+)c-kit(+)flt3(+) cells. Thus, whereas KL appears crucial for recruitment of FL + IL-7-unresponsive candidate (c-kit(+)flt3(-)) murine stem cells, FL is essential and sufficient for development toward lymphoid restricted progenitors from a population of (c-kit(+)flt3(+)) multipotent short-term reconstituting progenitors.     

Functional differences between CD38- and DR- subfractions of CD34+ bone marrow cells

Several studies have previously demonstrated enrichment in primitive progenitor cells in subfractions of CD34+ bone marrow (BM) cells not expressing CD38 or HLA-DR (DR) antigens. However, no studies have directly compared these two cell populations with regard to their content of primitive and more committed progenitor cells. Flow cytometric analysis of immunomagnetic isolated CD34+ cells demonstrated little overlap between CD34+CD38- and CD34+DR- progenitor subpopulations in that only 12% to 14% of total CD34+DR- and CD34+CD38- cells were double negative (CD34+CD38-DR-). Although the number of committed myeloid progenitor cells (colony-forming units granulocyte- macrophage) was reduced in both subpopulations, only CD34+CD38- cells were significantly depleted in committed erythroid progenitor cells (burst-forming units-erythroid). In single-cell assay, CD34+CD38- cells showed consistently poorer response to single as opposed to multiple hematopoietic growth factors as compared with unfractionated CD34+ cells, indicating that the CD34+CD38- subset is relatively enriched in primitive hematopoietic progenitor cells. Furthermore, CD34+CD38- and CD34+DR- cells, respectively, formed 3.2-fold and 1.6-fold more high proliferative potential colony-forming cell (HPP-CFC) colonies than did unfractionated CD34+ cells. Finally, CD34+CD38-DR- cells were depleted in HPP-CFCs as compared with CD34+CD38+DR+ cells. The results of the present study suggest that both the CD38- and DR- subfractions of CD34+ bone marrow cells are enriched in primitive hematopoietic progenitor cells, with the CD34+CD38- subpopulation being more highly enriched than CD34+DR- cells.     

Complement C3b receptors on erythrocytes in patients with juvenile rheumatoid arthritis

Forty patients with juvenile rheumatoid arthritis (JRA) were examined for C3b receptor (CR1) levels on erythrocytes (by an enzyme-linked immunosorbent assay), levels of circulating immune complexes (IC) (by a polyethylene glycol precipitation-complement consumption method), C3d split products (by intermediate gel rocket immunoelectrophoresis), and HLA-A, B, and C (by microdroplet lymphocytotoxicity test). Lower CR1 levels were found predominantly among patients with JRA (mean 57%) compared with 40 age-matched controls (mean 68%) (P = 0.008). The CR1 levels differed when the JRA patients were grouped by mode of disease onset and by clinical state at the time of testing (levels in patients with pauciarticular disease were higher than those in patients with polyarticular disease, and levels in patients with polyarticular disease were higher than those in patients with systemic disease) or by sex (girls had higher levels than boys; P = 0.01). The levels of circulating IC and C3d were elevated in 25% and 40% of JRA patients, respectively, and were mutually correlated (P less than 0.05, tau = 0.20). A negative correlation was found between levels of C3d and the numbers of CR1 (P less than 0.01, tau = -0.28), but concentrations of circulating IC did not correlate with CR1 values. CR1 levels were the same in 6 HLA-B27 positive and 25 HLA-B27 negative patients. These findings do not represent conclusive evidence that the number of CR1 on erythrocytes serves as a predictor of disease severity or as an indicator of disease activity in patients with JRA.     

Pretransplantation Exercise and Hematopoietic Cell Transplantation Survival: A Secondary Analysis of Blood and Marrow Transplant Clinical Trials Network (BMT CTN 0902)

Blood and Marrow Transplant Clinical Trials Network (BMT CTN) protocol 0902 evaluated whether exercise and stress management training before hematopoietic cell transplantation (HCT) improved physical and mental functioning after HCT. Neither overall survival nor other patient-reported transplantation outcomes were improved by the training intervention. In some animal studies of HCT, moderate-intensity exercise for 8 weeks before HCT has been associated with positive effects on hematopoietic progenitors, resulting in improved donor engraftment and improved survival. Accordingly, we performed a secondary analysis of data from BMT CTN 0902 to determine whether exercise engagement before HCT was associated with engraftment and survival. We found no significant associations between self-reported pre-HCT exercise levels and engraftment or survival. There was also no effect of pretransplantation exercise on either neutrophil or platelet engraftment. These findings do not support the observations in animal models but are limited by several shortcomings that do not refute the hypothesis that exercise before HCT may be beneficial.