Hypoxic adipocytes pattern early heterotopic bone formation

Abstract

The factors contributing to heterotopic ossification, the formation of bone in abnormal soft-tissue locations, are beginning to emerge, but little is known about microenvironmental conditions promoting this often devastating disease. Using a murine model in which endochondral bone formation is triggered in muscle by bone morphogenetic protein 2 (BMP2), we studied changes near the site of injection of BMP2-expressing cells. As early as 24 hours later, brown adipocytes began accumulating in the lesional area. These cells stained positively for pimonidazole and therefore generated hypoxic stress within the target tissue, a prerequisite for the differentiation of stem cells to chondrocytes and subsequent heterotopic bone formation. We propose that aberrant expression of BMPs in soft tissue stimulates production of brown adipocytes, which drive the early steps of heterotopic endochondral ossification by lowering oxygen tension in adjacent tissue, creating the correct environment for chondrogenesis. Results in misty gray lean mutant mice not producing brown fat suggest that white adipocytes convert into fat-oxidizing cells when brown adipocytes are unavailable, providing a compensatory mechanism for generation of a hypoxic microenvironment. Manipulation of the transcriptional control of adipocyte fate in local soft-tissue environments may offer a means to prevent or treat development of bone in extraskeletal sites.

Figure 1

Histological analysis of heterotopic bone formation stimulated by BMP2. a: Day 1, cells morphologically similar to brown adipocytes (arrows) detected between muscle fibers. b: Day 3 or 4, immature vessels (arrows) leaking red blood cells (inset, VWF staining). c: Day 5 or 6, cartilage matrix with embedded chondrocytes [arrows; inset, S-100 staining; muscle (M)]; d: Day 7, bone (B). Original magnifications: ×20.5 (a, c); ×10.5 (b, d).

Figure 2

a: Adipocytes recruited after Ad5F35-BMP2 and stained with Bodipy 493/502. b: Bodipy staining of muscle injected with cells carrying an empty vector, Ad5F35-null. c: Brown (yellow arrows) and white (white arrows) adipocytes after staining with Mitotracker. d: Mitotracker staining of muscle injected with cells carrying an empty vector. Tissues in a and c were harvested 24 hours after injection of BMP2-producing cells, and those in b and d, 24 hours after injection of cells transduced with empty vector. Original magnifications, ×20.5.

Figure 3

Multilocular adipocytes observed after injection of BMP2-producing cells express UCP-1, a unique marker of brown adipocytes. Staining with antibodies to UCP-1 detected the uncoupling protein (dark brown) on day 2 after stimulation with BMP2 in muscle from both NOD/SCID mice (A) and C57BL/6 mice (B) but not in muscle stimulated with a control vector (Ad5F35-HM4) (C) used to transduce MRC-5 cells injected into a NOD-SCID mouse or taken from a misty mouse after treatment with MC3T3 cells transduced to express BMP2 (D). Original magnifications: ×20.5 (A, D); ×40.5 (B, C).

Figure 4

Genes involved in energy metabolism and oxygen consumption are up-regulated. A: On days 1 and 2 after injection of BMP2-producing cells, microarray analysis was performed as described in the Materials and Methods section. The ratio of the amount of gene product expressed using BMP2-producing cells/the amount produced using cells transduced with empty vector is shown. B and C: Pathways involved in oxygen consumption. Glycolysis and the citric acid produce NADH, which is shuttled to the electron transport chain. NADH provides the proton motive force necessary for the production of ATP in coupled respiration and heat in uncoupled respiration. Both of these processes consume molecular oxygen, converting it to water. In brown adipocytes, UCP-1 is present converting the energy generated through the electron transport chain to heat.

Figure 5

Brown adipocytes generate hypoxia in tissue stimulated with BMP2. NOD/SCID mice were injected with BMP2-producing cells as described in Materials and Methods. A: Hypoxic regions appear in and near the site of injection. On day 3, mice were injected with pimonidazole, which was subsequently detected with an antibody against this compound (Hypoxyprobe monoclonal antibody). One of the hypoxic regions is shown in a. b: The growth plate of an uninfected NOD/SCID mouse processed similarly, and c: the lack of staining in the same region in a mouse 3 days after injection of cells transduced with control virus Ad5F35-null. Sections obtained at day 3 and embedded in paraffin were double-labeled with antibodies against pimonidazole (Hypoxyprobe; d, green) and PGC-1α (e, red). f: A merger of d and e. B: a: The close correlation between positive staining with the Hypoxyprobe monoclonal antibody (brown) and brown adipocytes 1 day after injection of BMP2-producing cells; b: a large portion of the area of the lesion is hypoxic 4 days of injection of BMP2-producing cells. c: Significant staining of cells in the area of injection for HIF-1, 2 days after injection. Original magnifications: ×10.5 (A, Bc); ×20.5 (Ba); ×4.5 (Bb).

Figure 6

Kinetics of UCP-1 synthesis in relation to hypoxia. Sections obtained either 1 (a), 2 (b), 4 (c), or 5 (d) days after injection of BMP2-producing cells were stained with a mixture of Hypoxyprobe mouse monoclonal antibody (red color) and a rabbit polyclonal antibody against UCP-1 (green color). Antibody binding was detected by incubation of the stained section with Qdot 525 goat F(ab′)₂ anti-rabbit IgG conjugate (red) and Q-dot 565 goat F(ab′)₂ anti-mouse IgG conjugate (green). Green arrows, cells expressing UCP-1; red arrows, hypoxic cells not expressing UCP-1; yellow arrows, hypoxic cells expressing UCP-1. Original magnifications, ×20.5.

Figure 7

Analysis of fat burning in misty mice that lack brown fat. BMP2-producing MC3T3 cells were injected into misty (n = 5) and C57BL/6 mice (n = 5), and tissue samples were examined for brown adipocytes on days 3, 4, and 6. Top left and right panels show the presence (C57BL/6) and absence (misty) of brown adipocytes between muscle fibers.

Figure 8

Analysis of hypoxia in misty and C57BL/6 mice. Subgroups of the mice described in Figure 7 were injected with pimonidazole at 3 days after injection and euthanized, and their limbs were prepared for staining with Hypoxyprobe. a: Day 4; c, day 3; and e, day 4; for C57BL/6 mice. b: Day 4; d, day 3; and f, day 4; for misty mice. Brown staining corresponds to areas of hypoxia.

Figure 9

Morphometric analysis of bone formation in misty and C57BL/6 mice. a and b: Micro-CT analysis of the bone formed at 14 days after injection with BMP2-producing MC3T3 cells in misty and C57BL/6 mice, with comparison of mineralized tissue volumes. c: Histology (H&E) of sections of bone in misty (top) and C57BL/6 mice. d: Example of sections analyzed by histomorphometry, (misty, top).