ABSTRACT
Hematopoietic stem cells (HSCs) are localized within specialized microenvironments throughout the BM. Nestin-expressing (Nestin(+)) mesenchymal stromal cells (MSCs) are important in the perivascular space. Rac is critical for MSC cell shape in vitro, whereas its function in MSCs in vivo remains poorly characterized. We hypothesized that deletion of Rac in the Nestin(+) cells would perturb the perivascular space, altering HSC localization and hematopoiesis. Nestin-Cre-directed excision of Rac1 in Rac3(-/-) mice reduces Nestin(+) cells in the marrow. We observed a 2.7-fold decrease in homing of labeled wild-type hematopoietic cells into Rac1(Δ/Δ)Rac3(-/-) mice compared with control mice. Rac1(Δ/Δ)Rac3(-/-) mice demonstrated a marked decrease in arterioles and an increase in the number and volume of venous sinusoids in the marrow that was associated with a reduction in the numbers of immunophenotypically and functionally-defined long-term HSCs in the marrow, a decrease in colony-forming cells and a reduction in circulating progenitors. Rac-deleted animals demonstrated a significant increase in trabecular bone. These data demonstrate that Rac GTPases play an important role in the integrity of perivascular space. Increased trabecular bone and sinusoidal space and decreased arteriolar volume in this model were associated with decreased HSC, underscoring the complexity of regulation of hematopoiesis in the perivascular space.
Subject(s)
Bone Marrow/metabolism , Bone Marrow/pathology , Hematopoiesis/genetics , rac GTP-Binding Proteins/genetics , Animals , Apoptosis/genetics , Blood Vessels , Bone and Bones/metabolism , Bone and Bones/pathology , Cellular Microenvironment , Chemokine CXCL12/genetics , Chemokine CXCL12/metabolism , Endothelial Cells/metabolism , Hematopoietic Stem Cells/metabolism , Immunophenotyping , Mice , Mice, Knockout , Nestin/genetics , Nestin/metabolism , Osteoblasts/metabolism , Stem Cell Factor/metabolism , rac GTP-Binding Proteins/deficiencyABSTRACT
Progenitor B cells reside in complex bone marrow (BM) microenvironments where they receive signals for growth and maturation. We reported previously that the CXCL12-focal adhesion kinase (FAK)-VLA4 pathway plays an important role in progenitor B cell adhesion and migration. In this study, we have conditionally targeted in B cells FAK, and found that the numbers of progenitor pro-B, pre-B, and immature B cells are reduced by 30-40% in B cell-specific FAK knockout mice. When cultured in methylcellulose with IL-7 ± CXCL12, Fak-deleted pro-B cells yield significantly fewer cells and colonies. Using in situ quantitative imaging cytometry, we establish that in longitudinal femoral BM sections, pro-B cells are preferentially localized in close proximity to the endosteum of the metaphyses and the diaphysis. Fak deletion disrupts the nonrandom distribution of pro-B cells and induces the mobilization of pro-B cells to the periphery in vivo. These effects of Fak deletion on pro-B cell mobilization and localization in BM are amplified under inflammatory stress, that is, after immunization with nitrophenol-conjugated chicken γ-globulin in alum. Collectively, these studies suggest the importance of FAK in regulating pro-B cell homeostasis and maintenance of their spatial distribution in BM niches.
Subject(s)
B-Lymphocytes/cytology , Bone Marrow/ultrastructure , Focal Adhesion Kinase 1/physiology , Hematopoietic Stem Cells/enzymology , Lymphopoiesis/physiology , Animals , Apoptosis , B-Lymphocytes/transplantation , Bone Marrow/immunology , Cells, Cultured/cytology , Cells, Cultured/drug effects , Cellular Microenvironment , Chemokine CXCL12/physiology , Chemotaxis, Leukocyte/physiology , Colony-Forming Units Assay , Female , Focal Adhesion Kinase 1/deficiency , Focal Adhesion Kinase 1/genetics , Hematopoietic Stem Cell Mobilization , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Homeostasis , Integrin alpha4beta1/physiology , Interleukin-7/pharmacology , Lymphopenia/etiology , Male , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
The existence of a haematopoietic stem cell niche as a spatially confined regulatory entity relies on the notion that haematopoietic stem and progenitor cells (HSPCs) are strategically positioned in unique bone marrow microenvironments with defined anatomical and functional features. Here, we employ a powerful imaging cytometry platform to perform a comprehensive quantitative analysis of HSPC distribution in bone marrow cavities of femoral bones. We find that HSPCs preferentially localize in endosteal zones, where most closely interact with sinusoidal and non-sinusoidal bone marrow microvessels, which form a distinctive circulatory system. In situ tissue analysis reveals that HSPCs exhibit a hypoxic profile, defined by strong retention of pimonidazole and expression of HIF-1α, regardless of localization throughout the bone marrow, adjacency to vascular structures or cell-cycle status. These studies argue that the characteristic hypoxic state of HSPCs is not solely the result of a minimally oxygenated niche but may be partially regulated by cell-specific mechanisms.