The vasculature of bone marrow differs from that in other organs, and its characteristics should be considered when exploring the medullar angiogenesis associated with hematological malignancies. We show here that the human bone marrow sinusoidal cell line HBME-1 has a specific expression pattern of angiogenic factors and receptors, characterized by a unique VEGFR3(+), Tie2(-) signature, that resembles the in vivo pattern. Moreover, the HBME-1 cultured for up to 3 days in hypoxic conditions, similar to those found in the bone marrow, specifically downregulated expression of VEGFR1, VEGFR2 and ETAR. Thus, a model using bone marrow sinusoidal cells cultured under reduced oxygen tension may be more relevant than classical in vitro endothelial cultures for understanding the interactions between endothelial and malignant cells in the medullar microenvironment.
[Show abstract][Hide abstract] ABSTRACT: The study of human microvascular endothelial cells has been limited, because these cells are difficult to isolate in pure culture, are fastidious in their in vitro growth requirements, and have a very limited lifespan. In order to overcome these difficulties, we have transfected human dermal microvascular endothelial cells (HMEC) with a PBR-322-based plasmid containing the coding region for the simian virus 40 A gene product, large T antigen, and succeeded in immortalizing them. These cells, termed CDC/EU.HMEC-1 (HMEC-1), have been passaged 95 times to date and show no signs of senescence, whereas normal microvascular endothelial cells undergo senescence at passages 8-10. HMEC-1 exhibit typical cobblestone morphology when grown in monolayer culture, express and secrete von Willebrand's Factor, take up acteylated low-density lipoprotein, and rapidly form tubes when cultured on matrigel. HMEC-1 grow to densities three to seven times higher than microvascular endothelial cells and require much less stringent growth medium. HMEC-1 will grow in the absence of human serum, whereas microvascular endothelial cells require culture medium supplemented with 30% human serum. These cells express other cell-surface molecules typically associated with endothelial cells, including CD31 and CD36 and epitopes identified by monoclonal antibodies EN4 and PAL-E. They also express the cell adhesion molecules ICAM-1 and CD44 and following stimulation with interferon-gamma express major histocompatibility complex class II antigens. HMEC-1 specifically bind lymphocytes in cell adhesion assays. Thus HMEC-1 is the first immortalized human microvascular endothelial cell line that retains the morphologic, phenotypic, and functional characteristics of normal human microvascular endothelial cells.
[Show abstract][Hide abstract] ABSTRACT: Myelosuppression damages the bone marrow (BM) vascular niche, but it is unclear how regeneration of bone marrow vessels contributes to engraftment of transplanted hematopoietic stem and progenitor cells (HSPCs) and restoration of hematopoiesis. We found that chemotherapy and sublethal irradiation induced minor regression of BM sinusoidal endothelial cells (SECs), while lethal irradiation induced severe regression of SECs and required BM transplantation (BMT) for regeneration. Within the BM, VEGFR2 expression specifically demarcated a continuous network of arterioles and SECs, with arterioles uniquely expressing Sca1 and SECs uniquely expressing VEGFR3. Conditional deletion of VEGFR2 in adult mice blocked regeneration of SECs in sublethally irradiated animals and prevented hematopoietic reconstitution. Similarly, inhibition of VEGFR2 signaling in lethally irradiated wild-type mice rescued with BMT severely impaired SEC reconstruction and prevented engraftment and reconstitution of HSPCs. Therefore, regeneration of SECs via VEGFR2 signaling is essential for engraftment of HSPCs and restoration of hematopoiesis.
[Show abstract][Hide abstract] ABSTRACT: The bone marrow contains a variety of blood vessels that have different functions in bone marrow maintenance and hematopoiesis. Arterioles control the flow of blood into bone marrow compartments, and sinusoids serve as a conduit to the bloodstream and as niches for megakaryocyte development. Most studies of bone marrow vasculature, including studies quantifying changes in the marrow vascular by microvascular density, do not differentiate between different types of marrow vessels. Recognizing changes in different types of blood vessels after chemotherapy exposure or during leukemia development has important physiological implications. We hypothesized that the functional heterogeneity of marrow vasculature could be recognized through the use of functional markers such as tyrosine kinase with immunoglobulin and epidermal growth factor homology domains-2 (Tie2) expression or 1,1-dioctadecyl -3,3,3,3-tetramethyl-indocarbocyanine perchlorate with acetylated low-density lipoprotein (DiI-Ac-LDL) uptake.
When transgenic mice with green fluorescent protein (GFP) expressed downstream of the Tie2 promoter were injected with Ac-LDL, Ac-LDL was specifically endocytosed by sinusoids, and Tie2 expression was more pronounced in the arteries, arterioles, and transitional capillaries. Combining these 2 functional endothelial markers and using confocal microscopy to obtain 3-dimensional images, we identified transitional zones where arterioles emptied into the sinusoids. Alternatively, coinjection of lectin with DiI-Ac-LDL has a similar result in normal mice, as seen in Tie2/GFP mice, and can be used to differentiate vessel types in nontransgenic mice.
These results demonstrate that bone marrow vasculature is functionally heterogeneous. Methods to study changes in the marrow vasculature using microvascular density or quantifying changes in the vascular niche need to take this heterogeneity into account.
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