Impact of Anti-Angiogenic Treatment on Bone Vascularization in a Murine Model of Breast Cancer Bone Metastasis Using Synchrotron Radiation Micro-CT.

Bone metastases are frequent complications of breast cancer, facilitating the development of anarchic vascularization and induce bone destruction. Therefore, anti-angiogenic drugs (AAD) have been tested as a therapeutic strategy for the treatment of breast cancer bone metastasis. However, the kinetics of skeletal vascularization in response to tumor invasion under AAD is still partially understood. Therefore, the aim of this study was to explore the effect of AAD on experimental bone metastasis by analyzing the three-dimensional (3D) bone vasculature during metastatic formation and progression. Seventy-three eight-week-old female mice were treated with AAD (bevacizumab, vatalanib, or a combination of both drugs) or the vehicle (placebo) one day after injection with breast cancer cells. Mice were sacrificed eight or 22 days after tumor cell inoculation (time points T1 and T2, respectively). Synchrotron radiation microcomputed tomography (SR-µCT) was used to image bone and blood vessels with a contrast agent. Hence, 3D-bone and vascular networks were simultaneously visualized and quantitatively analyzed. At T1, the trabecular bone volume fraction was significantly increased (p < 0.05) in the combined AAD-treatment group, compared to the placebo- and single AAD-treatment groups. At T2, only the bone vasculature was reduced in the combined AAD-treatment group (p < 0.05), as judged by measurement of the blood vessel thickness. Our data suggest that, at the early stage, combined AAD treatment dampens tumor-induced bone resorption with no detectable effects on bone vessel organization while, at a later stage, it affects the structure of bone microvascularization.

Parathyroid Hormone Remodels Bone Transitional Vessels and the Leptin Receptor-Positive Pericyte Network in Mice.

Intermittent parathyroid hormone (iPTH) is anti-osteoporotic and affects bone vessels. Transitional capillaries close to the bone surface, which express both endomucin (Edm) and CD31, bear leptin receptor-expressing (LepR) perivascular cells that may differentiate into osteoblasts. Increased numbers of type H endothelial cells (THEC; ie, Edmhi /CD31hi cells assessed by flow cytometry, FACS) are associated with higher bone formation in young mice. We hypothesized that iPTH administration impacts transitional vessels by expanding THECs. Four-month-old C57/Bl6J female mice were injected with PTH 1-84 (100 µg/kg/d) or saline (CT) for 7 or 14 days. We quantified LepR+ , CD31+ , Edm+ cells and THECs by FACS in hindlimb bone marrow, and Edm/LepR double immunolabelings on tibia cryosections. Additionally, we analyzed bone mRNA expression of 87 angiogenesis-related genes in mice treated with either intermittent or continuous PTH (iPTH/cPTH) or saline (CT) for 7, 14, and 28 days. iPTH dramatically decreased the percentage of THECs by 78% and 90% at days 7 and 14, respectively, and of LepR+ cells at day 14 (-46%) versus CT. Immunolabeling quantification showed that the intracortical Edm+ -vessel density increased at day 14 under iPTH. In the bone marrow, perivascular LepR+ cells, connected to each other via a dendrite network, were sparser under iPTH at day 14 (-58%) versus CT. iPTH decreased LepR+ cell coverage of transitional vessels only (-51%), whereas the number of LepR+ cells not attached to vessels increased in the endocortical area only (+ 49%). Transcriptomic analyses showed that iPTH consistently upregulated PEDF, Collagen-18α1, and TIMP-1 mRNA expression compared with CT and cPTH. Finally, iPTH increased immunolabeling of endostatin, a Collagen-18 domain that can be cleaved and become antiangiogenic, in both endocortical (79%) and peritrabecular transitional microvessels at day 14. Our results show that iPTH specifically remodels transitional vessels and suggest that it promotes LepR+ cell mobilization from these vessels close to the bone surface. © 2019 American Society for Bone and Mineral Research.

Bone Shaft Revascularization After Marrow Ablation Is Dramatically Accelerated in BSP-/- Mice, Along With Faster Hematopoietic Recolonization.

The bone organ integrates the activity of bone tissue, bone marrow, and blood vessels and the factors ensuring this coordination remain ill defined. Bone sialoprotein (BSP) is with osteopontin (OPN) a member of the small integrin binding ligand N-linked glycoprotein (SIBLING) family, involved in bone formation, hematopoiesis and angiogenesis. In rodents, bone marrow ablation induces a rapid formation of medullary bone which peaks by ∼8 days (d8) and is blunted in BSP-/- mice. We investigated the coordinate hematopoietic and vascular recolonization of the bone shaft after marrow ablation of 2 month old BSP+/+ and BSP-/- mice. At d3, the ablated area in BSP-/- femurs showed higher vessel density (×4) and vascular volume (×7) than BSP+/+. Vessel numbers in the shaft of ablated BSP+/+ mice reached BSP-/- values only by d8, but with a vascular volume which was twice the value in BSP-/-, reflecting smaller vessel size in ablated mutants. At d6, a much higher number of Lin- (×3) as well as LSK (Lin- IL-7Rα- Sca-1hi c-Kithi , ×2) and hematopoietic stem cells (HSC: Flt3- LSK, ×2) were counted in BSP-/- marrow, indicating a faster recolonization. However, the proportion of LSK and HSC within the Lin- was lower in BSP-/- and more differentiated stages were more abundant, as also observed in unablated bone, suggesting that hematopoietic differentiation is favored in the absence of BSP. Interestingly, unablated BSP-/- femur marrow also contains more blood vessels than BSP+/+, and in both intact and ablated shafts expression of VEGF and OPN are higher, and DMP1 lower in the mutants. In conclusion, bone marrow ablation in BSP-/- mice is followed by a faster vascular and hematopoietic recolonization, along with lower medullary bone formation. Thus, lack of BSP affects the interplay between hematopoiesis, angiogenesis, and osteogenesis, maybe in part through higher expression of VEGF and the angiogenic SIBLING, OPN. J. Cell. Physiol. 232: 2528-2537, 2017. © 2016 Wiley Periodicals, Inc.

Assessment of bone vascularization and its role in bone remodeling.

Bone is a composite organ that fulfils several interconnected functions, which may conflict with each other in pathological conditions. Bone vascularization is at the interface between these functions. The roles of bone vascularization are better documented in bone development, growth and modeling than in bone remodeling. However, every bone remodeling unit is associated with a capillary in both cortical and trabecular envelopes. Here we summarize the most recent data on vessel involvement in bone remodeling, and we present the characteristics of bone vascularization. Finally, we describe the various techniques used for bone vessel imaging and quantitative assessment, including histology, immunohistochemistry, microtomography and intravital microscopy. Studying the role of vascularization in adult bone should provide benefits for the understanding and treatment of metabolic bone diseases.

Parathyroid hormone 1-84 targets bone vascular structure and perfusion in mice: impacts of its administration regimen and of ovariectomy.

Bone vessel functions during bone remodeling are poorly understood. They depend on both vessel network structure and vasomotor regulation. Parathyroid hormone (PTH) is a systemic vasodilator that may modulate microvascularization. Moreover, although intermittent PTH is anti-osteoporotic, continuous PTH administration can be catabolic for bone. Finally, ovariectomy (OVX) reduces bone perfusion and vessel density in mice. We reasoned that the effects of PTH on bone vascularization might depend on its administration regimen and be impacted by ovariectomy. A 100-µg/kg PTH 1-84 daily dose was administered for 15 days to 4-month-old female C57BL/6 mice, either as daily sc injection (iPTH) or continuously (cPTH; ALZET minipump). Blood pressure (BP) and tibia bone perfusion were measured in vivo with a laser Doppler device. Histomorphometry of bone and barium-contrasted vascular network were performed on the same tibia. Compared with untreated controls, both iPTH and cPTH increased bone formation but had opposite effects on resorption. Both iPTH and cPTH were slightly angiogenic. Intermittent PTH increased microvessel size (+48%, p < 0.001), whereas cPTH decreased it (-29%, p = 0.009). iPTH increased bone perfusion (27%, p < 0.001) with no change in BP, whereas cPTH did not. The vascular effects of a 15-day iPTH treatment were analyzed in OVX mice and compared with sham-operated and OVX untreated controls. Two other anti-osteoporotic drugs, zoledronate (one injection, 70 µg/kg) and propranolol, (5 mg/kg/d) were tested in OVX mice. Although no change in bone mass was observed, iPTH stimulated bone formation and prevented the OVX-induced reduction in bone perfusion and vessel density. Both zoledronate and propranolol strongly lowered bone turnover, but surprisingly, zoledronate prevented OVX-induced reduction in bone perfusion but propranolol did not. Our integrative approach thus demonstrates that the effects of PTH on bone vessel structure and function depend on its mode of administration as well as on the HPG-axis hormonal status, and that OVX-induced vascular changes are prevented by iPTH.

Validated Laser Doppler protocol for measurement of mouse bone blood perfusion - response to age or ovariectomy differs with genetic background.

The physiological role of bone vascularization in bone metabolism begins to be understood; however, its involvement in pathological situations remains poorly explored. Bone blood supply depends on both vascular density and blood flow. However, in mice, the specific evaluation of perfusion in bone suffers from a lack of easy-handling measurement tools. In the present study, we first developed a Laser Doppler Perfusion Measurement (LDPM) protocol in mouse tibia, which we validated with ex vivo and in vivo experiments. Then we carried out a study associating both structural (vascular quantitative histomorphometry) and functional (LDPM) approaches. We studied the effects of aging in 4, 7 and 17 month-old male mice and the early effects of ovariectomy in 4 month-old females. Both studies were carried out in inbred mice (C57BL/6) and in mice of mixed background (129sv/CD1). The significant differences we observed between strains in unchallenged 4 month-old animals concerned both perfusion and vascular density and depended on gender. Additionally, the age-related bone loss observed in male mice was not temporally associated with vascular changes in either strain. Between 7 and 17 months, we did not find any decrease in bone vascular density or perfusion. In contrast, ovariectomy triggered early vascular structural and functional adaptations which differed between genetic backgrounds. We observed that bone vessel density did not generally account for bone perfusion levels. In conclusion, we describe here a LDPM-based experimental protocol which provides a reproducible quantitative evaluation of bone perfusion in mouse tibia, hence allowing intergroup comparisons. This integrative structural and functional approach of bone vascularization showed that bone vascular adaptation occurs during aging or after ovariectomy and is affected by the genetic background.

Structure and quantification of microvascularisation within mouse long bones: what and how should we measure?

Bone marrow vascularisation is involved in both remodeling and hematopoïesis. Challenged mouse models often require imaging and quantitative assessment of blood vessels and bone cell activities for a better understanding of the role of the vascular system. In this study we compared images of mouse hind limb long bone vascularisation after infusion of either barium sulfate or lead chromate-loaded silicon. The images were then analyzed through histology as well as low-resolution and synchrotron-radiation microtomography. We show that barium sulfate infusion provides the best vessel images and furthermore, that it is compatible with staining procedures used in bone histomorphometry and CD31 immunohistochemistry. Bone marrow vascularisation displays large structural and spatial distribution heterogeneity, including large lobular clusters of sinusoids and an unexpectedly substantial amount of capillaries in the adipocytes-rich distal third of the tibia. For an unbiased assessment of bone vascular development/changes, these features must be taken into account. We describe the conditions under which the quantification of microvascularisation on histological sections of barium-infused long bones is reproducible, as applied to seven-month-old male C57/Bl6J and mixed CD1/129Sv/J mice, and we propose a nomenclature for the histological parameters measured. Finally, we validate our technique by studying the effect of ovariectomy on mouse tibial vascular density.