Original Full Length ArticleMice with increased angiogenesis and osteogenesis due to conditional activation of HIF pathway in osteoblasts are protected from ovariectomy induced bone loss
Highlights
► Osteoporotic bones display reduced perfusion and numbers of capillaries. ► Bone formation and angiogenesis are coupled through HIF signaling in osteoblasts. ► Active HIF signaling pathway in osteoblasts prevents ovariectomy induced bone loss.
Introduction
Osteoporosis is a skeletal disease characterized by low bone mass and microarchitectural deterioration with a resulting increase in bone fragility and subsequent susceptibility to fractures [1]. The prevalence of osteoporosis increases with increasing age in both men and women with postmenopausal women as a very high risk group [2]. Although the primary cause of postmenopausal osteoporosis is estrogen deficiency [3], it is also characterized by a reduction in the number of sinusoidal and arterial capillaries in the bone marrow and reduced bone perfusion [4], [5], [6] suggesting a role of vascular component in the pathogenesis of postmenopausal osteoporosis. The skeleton is a highly vascularized tissue that receives 5–20% of cardiac output [7]. Developmental studies show that a close coupling between angiogenesis and osteogenesis is essential for normal bone formation [8]. Vascular invasion into cartilage anlagen is essential for long bone formation during endochondral ossification [9]. Moreover, disorders in bone vasculature can affect skeletal homeostasis. For example, impairment of angiogenesis decreases trabecular bone formation as well as the expansion of the hypertrophic zone into the growth plate [10], and surgical interruption of the blood supply to bone results in marked decrease in bone density and strength [11].
On molecular level, developing vasculature and its maintenance is guided by oxygen-gradient and hypoxia-inducible factors (HIFs) [12]. The HIF signaling pathway is also a central regulator of osteogenesis and angiogenesis coupling [13]. The HIF family consists of 3 α subunits, HIF1α, HIF2α, and HIF3α, which form a heterodimer complex with the HIF1β subunit. HIF1β is constitutively expressed whereas HIF-α is an oxygen-labile protein. The α-subunit has an oxygen-dependent degradation domain (ODD) which is recognized by prolyl hydroxylase domain (PHD) proteins. Under normoxic circumstances, PHD proteins hydroxylate HIFα and form a complex with von Hippel–Lindau protein (pVHL), a E3 ubiquitin ligase, that primes HIFα for proteosomal degradation [14]. During hypoxic conditions, prolyl hydroxylation of the α subunit is blocked and HIFα protein accumulates in cytoplasm. It then freely translocates to the nucleus where it heterodimerizes with the β subunit and initiates HIF-responsive genes transcription [15]. Absence of pVHL, and hence the lack of proteosomal degradation of HIFα leads to accumulation of HIFα in the cytoplasm [13], [16], [17] and represents a mechanism to activate HIFα signaling. In-vivo conditional activation of HIFα signaling pathway in osteoblasts by osteoblast-specific deletion of Vhl (ΔVHL) results not only in a dramatic increase in bone mass but also increases the vasculature in bone marrow cavity [13]. Conversely, mice lacking HIF-1α and/or HIF-2α in osteoblasts (ΔHIF) showed a significant decrease in bone volume as well as blood vessel formation [13]. These data reveal a role of HIF signaling in osteoblasts in maintaining bone homeostasis and also in promoting skeletal vascularization. The progressive increase in bone volume in ΔVHL mice results from increased numbers and activity of osteoblasts whereas osteopenia in the ΔHIF is associated with a significant decrease in osteoblast numbers and osteoid volume. However, disruption of Vhl in-vitro does not affect osteoblast proliferation or function indicating that HIF-1α regulates bone formation via cell nonautonomous mechanism. In fact, the amount of bone in these mutant mice is directly proportional to the amount of skeletal vascularity which is proportional to the increased secretion of VEGF by osteoblasts, a downstream target of HIFα signaling.
In this study, we examined whether the skeletal vascularization is decreased in ovariectomized (OVX) mice and whether increasing skeletal blood vessel formation by activation of HIF signaling in osteoblasts can prevent ovariectomy induced bone loss. To answer this question, we used ovariectomized mice with conditional activation of HIFα signaling pathway in osteoblasts by osteoblast-specific deletion of Vhl (ΔVhl) and compared their bone phenotype with normal OVX mice. We have found that skeletal vascularization and bone volume are indeed decreased in OVX mice and the bone loss can be prevented by upregulation of HIF signaling as in ΔVhl mice.
Section snippets
Animal model
All procedures involving mice were approved by the Shanghai Jiaotong University Animal Study Committee and were carried out in accordance with the guide for the humane use and care of laboratory animals. Mice with osteoblast-specific inactivation of Vhl (∆Vhl) were generated by intercrossing osteocalcin-Cre (OC-Cre) transgenic mice [18] with mice homozygous for floxed Vhl allele [19] (both mice kindly provided by Dr. Thomas L. Clemens, Department of Orthopaedic Surgery, Johns Hopkins University
Ovariectomy does not alter body weight in control or ∆Vhl mice
∆Vhl mice were similar in body weight to control littermates before ovariectomy. After the surgery, both control and ∆Vhl ovariectomized (OVX) mice trended to gain a modest weight compared to the respective sham operated group; however, the weight gains were not significantly different (Fig. 1A) indicating minimal stress to the animals due to surgery. Estrogen depletion by ovariectomy resulted in obvious atrophy of uterus in both control and ∆Vhl mice (Figs. 1B and C), and accordingly, the
Discussion
In adults, bone is continuously remodeled throughout life, removing damaged bone and replacing it with new bone and then reconstructing the bone material composition and architecture, to maintain bone strength [21]. During bone remodeling, a precise balance between osteoclastic bone resorption and osteoblastic bone formation is essential in order to maintain the integrity of the bone. Any imbalance in the equilibrium alters bone remodeling that results in osteopetrosis or osteoporosis [22].
Conclusion
Estrogen deficiency induces bone loss and is associated with decreased skeletal vascularization. Here we show that the bones of ovariectomized mice have decreased expression of HIFα and pro-angiogenic factor VEGF leading to decreased bone vasculature along with bone volume. Maintenance of skeletal vasculature by upregulation of HIFα signaling in osteoblasts prevents the estrogen deficiency induced bone loss.
Acknowledgments
The authors thank Thomas L. Clemens for sharing HIF degrading protein von Hippel-Lindau mice. This work was supported by the National Natural Science Foundation of China (Grant No. 30872641), and the Science and Technology Commission of Shanghai Municipality (Grant No. 09DZ2200500).
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These authors contributed equally to this study.