High levels of acetylated low-density lipoprotein uptake and low tyrosine kinase with immunoglobulin and epidermal growth factor homology domains-2 (Tie2) promoter activity distinguish sinusoids from other vessel types in murine bone marrow

Abstract

Background: 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.

Methods and results: 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.

Conclusions: 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.

Figure 1. Comparison of in vitro and in vivo labeling of bone marrow using DiI-Ac-LDL by flow cytometry (n=3)

A. In vitro labeling. DiI-Ac-LDL was added to the flushed bone marrow cells in culture. Two DiI-Ac-LDL positive cell populations are present; the DiI-Ac-LDL low and the DiI-Ac-LDL high populations. We present the percentage of cells expressing lineage specific marker that have or have not endocytosed DiI-Ac-LDL. B. In vivo labeling. DiI-Ac-LDL was injected retro-orbitally into the mouse blood stream. Very few DiI-Ac-LDL positive cells were detected. Some positive cells were dead, auto-fluorescent cells. They appeared in the diagonal position in the unstained sample.

Figure 2. Injected DiI-Ac-LDL specifically marked the venular side of the murine marrow vasculature, including the marrow sinusoids (n=5–10)

A. A flushed bone marrow from the tibia of a C57BL/6 mouse that was injected with DiI-Ac-LDL demonstrates the sinusoidal network throughout the marrow (Scale bar = 600um). B. Confocal image of the network of sinusoids from a mouse bone marrow injected with DiI-Ac-LDL (Scale bar =150um). C. A section of the bone marrow of a GFP transgenic mouse that was injected with DiI-Ac-LDL shows the specificity of the DiI-Ac-LDL marker for the sinusoids (Scale bar = 50um). D. A section of bone marrow 4 days after receiving 950 cGy radiation. Vastly dilated sinusoids have endocytosed DiI-Ac-LDL. The individual sinusoidal endothelial cells can be identified (white arrows). Because of intramedullary hemorrhage, the dye has been endocytosed by other cells such as macrophages (yellow arrows). (Scale bar = 40um). E. A section from a mouse 9 days post-transplant from a GFP donor showing sinusoids were the only vessel that have endocytosed DiI-Ac-LDL (white arrows). A straight arteriole (based on the line of elongated nuclei (pink arrows) was DiI-Ac-LDL negative (Scale bar = 30um). F. Cells lining the central sinus also endocytose DiI-Ac-LDL (Scale bar =120um).

Figure 3. Tie2 promoter activity in different types of bone marrow blood vessels in Tie2/GFP transgenic mice (n=5–10)

A. Tie-2/GFP marrow core, viewed directly under the confocal microscope demonstrated the tree like structure of the arterial network (Scale bar = 300um). B. A bone marrow section from a Tie-2/GFP mouse that was injected with DiI-Ac-LDL shows GFP expression in an arteriole surrounded by smooth muscle nuclei (pink arrows). Sinusoids that endocytosed DiI-Ac-LDL did not express GFP (yellow arrows) (Scale bar = 25um). C. GFP expression in different types of vessels. The arteriole with smooth muscle surrounding (pink arrows) expressed GFP strongly. The transitional vessels (orange arrow) expressed less GFP in the vessel wall than around the nuclei. Trace amount of DiI-Ac-LDL can be seen inside the vessels. Capillaries (blue arrows) with very small diameter expressed GFP strongly, and trace amounts of DiI-Ac-LDL can also be see in the vessel (Scale bar =25um). D and E. DiI-Ac-LDL marked individual sinusoidal tubes. GFP expression in the sinusoid was localized to the perinuclear region (yellow arrows). (Scale bar = 25um). F. A sectioned bone marrow sample showed two sinusoids (yellow arrows) labeled by the DiI-Ac-LDL but with no GFP signal (Scale bar =20um).

Figure 4. Transitional zone from straight capillaries to the sinusoids (n=3)

This figure shows an example of the transitional zone in the marrow of a Tie-2/GFP transgenic mouse that was injected with DiI-Ac-LDL. The blue arrows point to a section of an arteriole characterized by the strong GFP expression in the vessel wall with relatively wide diameter. The small white arrows in the surrounding panels point to the straight capillaries that are 4–5 um in diameter. The large white arrows point to the transition vessels that are continuous with the straight capillaries but are wider in diameter and have relatively weaker GFP expression in the vessel wall compared to the strong expression from the perinuclear area. The pink arrows point to locations where transitional vessels empty into sinusoids, in which GFP expression is only present in the perinuclear area. The sinusoidal vessel walls are intensely labeled by DiI-Ac-LDL. (Scale bar = 100um).

Figure 5. Tie2 and DiI-Ac-LDL positive vessels in the metaphyseal area in Tie2/GFP mice (n=3)

Labeled “Ep Disk” on the top left corner in A refers to the epiphyseal disk area. “Trab Bone” in B refers to the trabecular bone. White arrows point to the possible transition zones in both A and B based on the yellow color of the nuclei of the endothelial cells because of the strong GFP expression in the perinuclear region and strong uptake DiI-Ac-LDL, a characteristic of transition vessels (see Figure 4). (Scale bar = 100 um in A and 40 um in B).

Figure 6. Lectin co-injection with DiI-Ac-LDL in differentiating vessels types in normal mice (n=3)

Green arrows in A demonstrate lectin labeled arterioles with lacking DiI-Ac-LDL staining, while the red arrow indicates a sinusoid labeled by both DiI-Ac-LDL and lectin fluorescing yellow. The pink arrows in both A and B point to non-endothelial cells that were labeled lectin that had leaked from the vessels. B, An example of transition vessel that was labeled by lectin. Short white arrow demonstrate the thin straight capillaries and the long white arrow indicates the widened transition vessels that were the continuation from the straight capillaries and emptied into the sinusoids. (Scale bar = 30um in A and 20um in B)

Figure 7. An illustration of the characteristics and the distributions of different types of vessels in the bone marrow

Green: Tie2 expression of the endothelial cells. In the arteries, arterioles and capillaries, Tie2/GFP is expressed in the whole cell including the cytoplasm. In the transition vessels and the sinusoids, GFP signal is mainly in the perinuclear area (small green circles). Red: DiI-Ac-LDL is endocytosed mainly by the sinusoidal endothelial cells. The transitional zone where the arterial system connects with the venular system is indicated by the large black circles.