The role of bone marrow (BM)-derived precursor cells in tumor angiogenesis is not known. We demonstrate here that tumor angiogenesis is associated with recruitment of hematopoietic and circulating endothelial precursor cells (CEPs). We used the angiogenic defective, tumor resistant Id-mutant mice to show that transplantation of wild-type BM or vascular endothelial growth factor (VEGF)-mobilized stem cells restore tumor angiogenesis and growth. We detected donor-derived CEPs throughout the neovessels of tumors and Matrigel-plugs in an Id1+/-Id3-/- host, which were associated with VEGF-receptor-1-positive (VEGFR1+) myeloid cells. The angiogenic defect in Id-mutant mice was due to impaired VEGF-driven mobilization of VEGFR2+ CEPs and impaired proliferation and incorporation of VEGFR1+ cells. Although targeting of either VEGFR1 or VEGFR2 alone partially blocks the growth of tumors, inhibition of both VEGFR1 and VEGFR2 was necessary to completely ablate tumor growth. These data demonstrate that recruitment of VEGF-responsive BM-derived precursors is necessary and sufficient for tumor angiogenesis and suggest new clinical strategies to block tumor growth.

Stem cells within the bone marrow (BM) exist in a quiescent state or are instructed to differentiate and mobilize to circulation following specific signals. Matrix metalloproteinase-9 (MMP-9), induced in BM cells, releases soluble Kit-ligand (sKitL), permitting the transfer of endothelial and hematopoietic stem cells (HSCs) from the quiescent to proliferative niche. BM ablation induces SDF-1, which upregulates MMP-9 expression, and causes shedding of sKitL and recruitment of c-Kit + stem/progenitors. In MMP-9 −/− mice, release of sKitL and HSC motility are impaired, resulting in failure of hematopoietic recovery and increased mortality, while exogenous sKitL restores hematopoiesis and survival after BM ablation. Release of sKitL by MMP-9 enables BM repopulating cells to translocate to a permissive vascular niche favoring differentiation and reconstitution of the stem/progenitor cell pool.

The US National Institute of Neurological Disorders and Stroke convened major stakeholders in June 2012 to discuss how to improve the methodological reporting of animal studies in grant applications and publications. The main workshop recommendation is that at a minimum studies should report on sample-size estimation, whether and how animals were randomized, whether investigators were blind to the treatment, and the handling of data. We recognize that achieving a meaningful improvement in the quality of reporting will require a concerted effort by investigators, reviewers, funding agencies and journal editors. Requiring better reporting of animal studies will raise awareness of the importance of rigorous study design to accelerate scientific progress.

Bronchoalveolar lavage is a powerful technique for sampling the epithelial lining fluid (ELF) of the lower respiratory tract but also results in a significant dilution of that fluid. To quantify the apparent volume of ELF obtained by bronchoalveolar lavage, urea was used as an endogenous marker of ELF dilution. Since urea diffuses readily through the body, plasma and in situ ELF urea concentrations are identical; thus ELF volume can be calculated using simple dilution principles. Using this approach, we determined that with a standard lavage procedure, the volume of ELF recovered from a normal human is 1.0 +/- 0.1 ml/100 ml of recovered lavage fluid. Time course experiments in which the saline used for lavage was permitted to remain in the lower respiratory tract for various "dwell times" suggested that diffusion of urea from sources other than recovered ELF can contribute to the total urea recovered resulting in an overestimate of the volume of ELF recovered. Thus, while reasonably accurate, the volume of ELF determined by urea must be considered an overestimate, or "apparent" volume. The ELF albumin concentration based on the apparent ELF volume was 3.7 +/- 0.3 mg/ml, a value that is in good agreement with direct measurements made by other techniques in experimental animals. The density of all inflammatory and immune effector cells on the epithelial surface of the lower respiratory tract, based on the apparent ELF volume, was 21,000 +/- 3,000 cells/microliter, a value that is twofold greater than that in blood.(ABSTRACT TRUNCATED AT 250 WORDS)

The molecular pathways involved in the differentiation of hematopoietic progenitors are unknown. Here we report that chemokine-mediated interactions of megakaryocyte progenitors with sinusoidal bone marrow endothelial cells (BMECs) promote thrombopoietin (TPO)-independent platelet production. Megakaryocyte-active cytokines, including interleukin-6 (IL-6) and IL-11, did not induce platelet production in thrombocytopenic, TPO-deficient (Thpo(-/-)) or TPO receptor-deficient (Mpl(-/-)) mice. In contrast, megakaryocyte-active chemokines, including stromal-derived factor-1 (SDF-1) and fibroblast growth factor-4 (FGF-4), restored thrombopoiesis in Thpo(-/-) and Mpl(-/-) mice. FGF-4 and SDF-1 enhanced vascular cell adhesion molecule-1 (VCAM-1)- and very late antigen-4 (VLA-4)-mediated localization of CXCR4(+) megakaryocyte progenitors to the vascular niche, promoting survival, maturation and platelet release. Disruption of the vascular niche or interference with megakaryocyte motility inhibited thrombopoiesis under physiological conditions and after myelosuppression. SDF-1 and FGF-4 diminished thrombocytopenia after myelosuppression. These data suggest that TPO supports progenitor cell expansion, whereas chemokine-mediated interaction of progenitors with the bone marrow vascular niche allows the progenitors to relocate to a microenvironment that is permissive and instructive for megakaryocyte maturation and thrombopoiesis. Progenitor-active chemokines offer a new strategy to restore hematopoiesis in a clinical setting.

The mechanisms through which hematopoietic cytokines accelerate revascularization are unknown. Here, we show that the magnitude of cytokine-mediated release of SDF-1 from platelets and the recruitment of nonendothelial CXCR4 + VEGFR1 + hematopoietic progenitors, 'hemangiocytes,' constitute the major determinant of revascularization. Soluble Kit-ligand (sKitL), thrombopoietin (TPO, encoded by Thpo) and, to a lesser extent, erythropoietin (EPO) and granulocyte-macrophage colony-stimulating factor (GM-CSF) induced the release of SDF-1 from platelets, enhancing neovascularization through mobilization of CXCR4 + VEGFR1 + hemangiocytes. Although revascularization of ischemic hindlimbs was partially diminished in mice deficient in both GM-CSF and G-CSF (Csf2 −/− Csf3 −/− ), profound impairment in neovascularization was detected in sKitLdeficient Mmp9 −/− as well as thrombocytopenic Thpo −/− and TPO receptor-deficient (Mpl −/− ) mice. SDF-1-mediated mobilization and incorporation of hemangiocytes into ischemic limbs were impaired in Thpo −/− , Mpl −/− and Mmp9 −/− mice. Transplantation of CXCR4 + VEGFR1 + hemangiocytes into Mmp9 −/− mice restored revascularization, whereas inhibition of CXCR4

Enough information has been gained from clinical trials to allow the conclusion that human gene transfer is feasible, can evoke biologic responses that are relevant to human disease, and can provide important insights into human biology. Adverse events have been uncommon and have been related to the gene delivery strategies, not to the genetic material being transferred. Human gene transfer still faces significant hurdles before it becomes an established therapeutic strategy. However, its accomplishments to date are impressive, and the logic of the potential usefulness of this clinical paradigm continues to be compelling.

The epithelial cells on the alveolar surface of the human lower respiratory tract are vulnerable to toxic oxidants derived from inhaled pollutants or inflammatory cells. Although these lung cells have intracellular antioxidants, these defenses may be insufficient to protect the epithelial surface against oxidants present at the alveolar surface. This study demonstrates that the epithelial lining fluid (ELF) of the lower respiratory tract contains large amounts of the sulfhydryl-containing antioxidant glutathione (GSH). The total glutathione (the reduced form GSH and the disulfide GSSG) concentration of normal ELF was 140-fold higher than that in plasma of the same individuals, and 96% of the glutathione in ELF was in the reduced form. Compared with nonsmokers, cigarette smokers had 80% higher levels of ELF total glutathione, 98% of which was in the reduced form. Studies of cultured lung epithelial cells and fibroblasts demonstrated that these concentrations of reduced glutathione were sufficient to protect these cells against the burden of H2O2 in the range released by alveolar macrophages removed from the lower respiratory tract of nonsmokers and smokers, respectively, suggesting that the glutathione present in the alveolar ELF of normal individuals likely contributes to the protective screen against oxidants in the extracellular milieu of the lower respiratory tract.