Involvement of Siglec-15 in regulating RAP1/RAC signaling in cytoskeletal remodeling in osteoclasts mediated by macrophage colony-stimulating factor.

DNAX-associated protein 12 kD size (DAP12) is a dominant immunoreceptor tyrosine-based activation motif (ITAM)-signaling adaptor that activates costimulatory signals essential for osteoclastogenesis. Although several DAP12-associated receptors (DARs) have been identified in osteoclasts, including triggering receptor expressed on myeloid cells 2 (TREM-2), C-type lectin member 5 A (CLEC5A), and sialic acid-binding Ig-like lectin (Siglec)-15, their precise role in the development of osteoclasts and bone remodeling remain poorly understood. In this study, mice deficient in Trem-2, Clec5a, Siglec-15 were generated. In addition, mice double deficient in these DAR genes and FcεRI gamma chain (FcR)γ, an alternative ITAM adaptor to DAP12, were generated. Bone mass analysis was conducted on all mice. Notably, Siglec-15 deficient mice and Siglec-15/FcRγ double deficient mice exhibited mild and severe osteopetrosis respectively. In contrast, other DAR deficient mice showed normal bone phenotype. Likewise, osteoclasts from Siglec-15 deficient mice failed to form an actin ring, suggesting that Siglec-15 promotes bone resorption principally by modulating the cytoskeletal organization of osteoclasts. Furthermore, biochemical analysis revealed that Sigelc-15 activates macrophage colony-stimulating factor (M-CSF)-induced Ras-associated protein-1 (RAP1)/Ras-related C3 botulinum toxin substrate 1 (Rac1) pathway through formation of a complex with p130CAS and CrkII, leading to cytoskeletal remodeling of osteoclasts. Our data provide genetic and biochemical evidence that Siglec-15 facilitates M-CSF-induced cytoskeletal remodeling of the osteoclasts.

Regional difference in the distribution of alkaline phosphatase, PHOSPHO1, and calcein labeling in the femoral metaphyseal trabeculae in parathyroid hormone-administered mice.

OBJECTIVES: This study aimed to elucidate whether the administration of parathyroid hormone (PTH) results in remodeling- or modeling-based bone formation in different regions of the murine femora, and whether the PTH-driven bone formation would facilitate osteoblastic differentiation into osteocytes. METHODS: Six-week-old male C57BL/6J mice were employed to examine the distribution of alkaline phosphatase (ALP), PHOSPHO1, podoplanin, and calcein labeling in two distinct long bone regions: the metaphyseal trabeculae close to the chondro-osseous junction (COJ) and those distant from the COJ in three mouse groups, a control group receiving a vehicle (Sham group) and groups receiving hPTH (1-34) twice a day (PTH BID group) or four times a day (PTH QID group) for two weeks. RESULTS: The Sham group showed PHOSPHO1-reactive mature osteoblasts localized primarily at the COJ, whereas the PTH BID/QID groups exhibited extended lines of PHOSPHO1-reactive osteoblasts even in regions distant from the COJ. The PTH QID group displayed fragmented calcein labeling in trabeculae close to the COJ, whereas continuous labeling was observed in trabeculae distant from the COJ. Osteoblasts tended to express podoplanin and PHOSPHO1 independently in the close and distant regions of the Sham group, while osteoblasts in the PTH-administered groups showed immunoreactivity of podoplanin and PHOSPHO1 together in the close and distant regions. CONCLUSIONS: Administration of PTH may accelerate remodeling-based bone formation in regions close to the COJ while predominantly inducing modeling-based bone formation in distant regions. PTH appeared to simultaneously facilitate osteoblastic bone mineralization and differentiation into osteocytes in both remodeling- and modeling-based bone formation.

Immunohistochemical and Morphometric Assessment on the Biological Function and Vascular Endothelial Cells in the Initial Process of Cortical Porosity in Mice With PTH Administration.

To clarify the cellular mechanism of cortical porosity induced by intermittent parathyroid hormone (PTH) administration, we examined the femoral cortical bone of mice that received 40 µg/kg/day (four times a day) human PTH (hPTH) (1-34). The PTH-driven cortical porosity initiated from the metaphyseal region and chronologically expanded toward the diaphysis. Alkaline phosphatase (ALP)-positive osteoblasts in the control mice covered the cortical surface, and endomucin-positive blood vessels were distant from these osteoblasts. In PTH-administered mice, endomucin-reactive blood vessels with TRAP-positive penetrated the ALP-positive osteoblast layer, invading the cortical bone. Statistically, the distance between endomucin-positive blood vessels and the cortical bone surface abated after PTH administration. Transmission electron microscopic observation demonstrated that vascular endothelial cells often pass through the flattened osteoblast layer and accompanied osteoclasts in the deep region of the cortical bone. The cell layers covering mature osteoblasts thickened with PTH administration and exhibited ALP, α-smooth muscle actin (αSMA), vascular cell adhesion molecule-1 (VCAM1), and receptor activator of NF-κB ligand (RANKL). Within these cell layers, osteoclasts were found near endomucin-reactive blood vessels. In PTH-administered femora, osteocytes secreted Dkk1, a Wnt inhibitor that affects angiogenesis, and blood vessels exhibited plasmalemma vesicle-associated protein, an angiogenic molecule. In summary, endomucin-positive blood vessels, when accompanied by osteoclasts in the ALP/αSMA/VCAM1/RANKL-reactive osteoblastic cell layers, invade the cortical bone, potentially due to the action of osteocyte-derived molecules such as DKK1.

Eldecalcitol Induces Minimodeling-Based Bone Formation and Inhibits Sclerostin Synthesis Preferentially in the Epiphyses Rather than the Metaphyses of the Long Bones in Rats.

This study aimed to examine minimodeling-based bone formation between the epiphyses and metaphyses of the long bones of eldecalcitol (ELD)-administered ovariectomized rats. Sixteen-week-old female rats were divided into four groups: sham-operated rats receiving vehicle (Sham group), ovariectomized (OVX) rats receiving vehicle (Vehicle group), or ELDs (30 or 90 ng/kg BW, respectively; ELD30 and ELD90 groups). ELD administration increased bone volume and trabecular thickness, reducing the number of osteoclasts in both the epiphyses and metaphyses of OVX rats. The Sham and Vehicle groups exhibited mainly remodeling-based bone formation in both regions. The epiphyses of the ELD groups showed a significantly higher frequency of minimodeling-based bone formation than remodeling-based bone formation. In contrast, the metaphyses exhibited significantly more minimodeling-based bone formation in the ELD90 group compared with the ELD30 group. However, there was no significant difference between minimodeling-based bone formation and remodeling-based bone formation in the ELD90 group. While the minimodeling-induced new bone contained few sclerostin-immunoreactive osteocytes, the underlying pre-existing bone harbored many. The percentage of sclerostin-positive osteocytes was significantly reduced in the minimodeling-induced bone in the epiphyses but not in the metaphyses of the ELD groups. Thus, it seems likely that ELD could induce minimodeling-based bone formation in the epiphyses rather than in the metaphyses, and that ELD-driven minimodeling may be associated with the inhibition of sclerostin synthesis.

Abaloparatide promotes bone repair of vertebral defects in ovariectomized rats by increasing bone formation.

Osteoporotic vertebral fracture (OVF) is the most common type of osteoporotic fracture and is associated with immobility and mortality. Bone anabolic agents, such as abaloparatide (ABL), are usually administered to patients with OVF to prevent subsequent fractures. Although several studies have shown that bone anabolic agents promote healing of long bone fractures, there is little evidence of their healing effect on vertebral bone fractures. In the present study, we investigated the effect of ABL on vertebral bone defects using ovariectomized (OVX) rats with vertebral body drill-hole defects, an animal model of OVF. Eight-week-old female Sprague-Dawley rats were subjected to OVX, followed by the 32-36 days of bone depletion period, once-daily subcutaneous ABL was administered to OVX rats at a dose of 30 µg/kg for a maximum of 6 weeks from the day of the vertebral defect surgery. We found that ABL significantly increased bone mineral content and improved trabecular structural parameters at the vertebral defect site. Moreover, ABL significantly increased bone strength of the defected vertebrae. Bone histochemical analysis revealed formation of thick trabecular bone networks at the defect site after ABL administration, consistent with an improvement in trabecular structural parameters by ABL. ABL increased ALPase- and PHOSPHO1-positive osteoblastic cells and ALPase/PCNA double-positive cells, indicating enhanced preosteoblast proliferation as well as bone formation at the defect site. On the other hand, ABL did not affect the number of cathepsin K-positive osteoclasts per bone surface, suggesting that ABL did not promote excessive bone resorption. Our findings suggest that ABL is useful not only for preventing secondary vertebral fractures but also for promoting bone healing in OVF.

Phosphorylated pullulan promotes calcification during bone regeneration in the bone defects of rat tibiae.

The current study aimed to evaluate bone tissue regeneration using a combination of ß-tricalcium phosphate (ßTCP) and phosphorylated pullulan (PPL, a phosphate-rich polysaccharide polymer consisting of maltotriose units). Round defects of 2 mm diameter were created in the arterial center of rat tibiae, which were further treated with vehicle (control group), ßTCP (ßTCP group), or ßTCP + PPL (ßTCP + PPL group) grafts. The control specimens without bone grafts exhibited rapid bone formation after 1 week; however, the regenerated bone was not resorbed until 4 weeks. In contrast, ßTCP-grafted specimens exhibited fewer but thicker trabeculae, whereas the ßTCP + PPL group displayed many fine trabeculae at 4 weeks. In the ßTCP + PPL group, new bone was associated with the ßTCP granules and PPL. Similarly, PHOSPHO1-positive osteoblasts were localized on the ßTCP granules as well as the PPL. On the other hand, TRAP-reactive osteoclasts predominantly localized on newly-formed bone and ßTCP granules rather than on the PPL. No significant differences were observed in the expression of Alp, Integrin αv, Osteopontin, Osteocalcin, and Dmp-1 in PPL-treated MC3T3-E1 osteoblastic cells, suggesting that PPL did not facilitate osteoblastic differentiation. However, von Kossa staining identified abundant needle-like calcified structures extending inside the PPL. Furthermore, transmission electron microscopy (TEM) revealed many globular structures identical to calcified nodules. In addition, calcified collagen fibrils were observed in the superficial layer of the PPL. Thus, PPL may serve as a scaffold for osteoblastic bone formation and promotes calcification on its surface. In conclusion, we speculated that ßTCP and PPL might promote bone regeneration and could be integrated into promising osteoconductive materials.

Early gene expression profiles of anabolic and catabolic molecules in murine bone after a single PTH injection.

The current study examined the gene expression profiles of anabolic and catabolic molecules after a single parathyroid hormone (PTH) injection in mice. No significant changes were observed in alkaline phosphatase area/tissue volume, tartrate-resistant acid phosphatase-positive osteoclasts, or static bone histomorphometry parameters. However, a sudden and significant decrease in Runx2 expression occurred at 1.5 h post-injection followed by immediate elevation, while sclerostin level was initially downregulated but gradually recovered. Meanwhile, Rankl expression initially increased and then returned to baseline. The prolonged elevation of anabolic molecules and transient increase in catabolic molecules may contribute to the anabolic effect of PTH treatment.

Differential osteoblastic activity in primary metaphyseal trabecular and secondary trabeculae of c-fos deficient mice.

OBJECTIVES: It has been highlighted that osteoblastic activities in remodeling-based bone formation are coupled with osteoclastic bone resorption while those in modeling-based bone formation are independent of osteoclasts. This study aimed to verify whether modeling-based bone formation can occur in the absence of osteoclasts. METHODS: We performed histochemical analyses on the bone of eight-week-old male wild-type and c-fos-/- mice. Histochemical analyses were conducted on primary trabeculae near the chondro-osseous junction (COJ), sites of modeling-based bone formation, and secondary trabeculae, sites of remodeling-based bone formation, in the femora and tibiae of mice. RESULTS: Alkaline phosphatase (ALP) immunoreactivity, a marker of osteoblastic lineages, was observed in the metaphyseal trabeculae of wild-type mice, while ALP was scattered throughout the femora of c-fos-/- mice. PHOSPHO1, an enzyme involved in matrix vesicle-mediated mineralization, was predominantly detected in primary trabeculae and also within short lines of osteoblasts in secondary trabeculae of wild-type mice. In contrast, femora of c-fos-/- mice showed several patches of PHOSPHO1 positivity in the primary trabeculae, but there were hardly any patches of PHOSPHO1 in secondary trabeculae. Calcein labeling was consistently observed in primary trabeculae close to the COJ in both wild-type and c-fos-/- mice; however, calcein labeling in the secondary trabeculae was only detected in wild-type mice. Transmission electron microscopic examination demonstrated abundant rough endoplasmic reticulum in the osteoblasts in secondary trabeculae of wild-type mice, but not in those of c-fos-/- mice. CONCLUSIONS: Osteoblastic activities at the sites of modeling-based bone formation may be maintained in the absence of osteoclasts.

Immunolocalization of Enzymes/Membrane Transporters Related to Bone Mineralization in the Metaphyses of the Long Bones of Parathyroid-Hormone-Administered Mice.

The present study aimed to demonstrate the immunolocalization and/or gene expressions of the enzymes and membrane transporters involved in bone mineralization after the intermittent administration of parathyroid hormone (PTH). The study especially focused on TNALP, ENPP1, and PHOSPHO1, which are involved in matrix vesicle-mediated mineralization, as well as PHEX and the SIBLING family, which regulate mineralization deep inside bone. Six-week-old male mice were subcutaneously injected with 20 µg/kg/day of human PTH (1-34) two times per day (n = 6) or four times per day (n = 6) for two weeks. Additionally, control mice (n = 6) received a vehicle. Consistently with an increase in the volume of the femoral trabeculae, the mineral appositional rate increased after PTH administration. The areas positive for PHOSPHO1, TNALP, and ENPP1 in the femoral metaphyses expanded, and the gene expressions assessed by real-time PCR were elevated in PTH-administered specimens when compared with the findings in control specimens. The immunoreactivity and/or gene expressions of PHEX and the SIBLING family (MEPE, osteopontin, and DMP1) significantly increased after PTH administration. For example, MEPE immunoreactivity was evident in some osteocytes in PTH-administered specimens but was hardly observed in control specimens. In contrast, mRNA encoding cathepsin B was significantly reduced. Therefore, the bone matrix deep inside might be further mineralized by PHEX/SIBLING family after PTH administration. In summary, it is likely that PTH accelerates mineralization to maintain a balance with elevated matrix synthesis, presumably by mediating TNALP/ENPP1 cooperation and stimulating PHEX/SIBLING family expression.

Histochemical assessment of osteoclast-like giant cells in Rankl-/- mice.

OBJECTIVES: We examined mice with gene deletion of Receptor activator of nuclear factor-κB (Rank) ligand (Rankl) to histologically clarify whether they contained progenitor cells committed to osteoclastic differentiation up to the stage requiring RANK/RANKL signaling. METHODS: The tibiae and femora of ten-week-old male wild-type, c-fos-/-, and Rankl-/- mice were used for immunohistochemistry and transmission electron microscopy (TEM). RESULTS: In Rankl-/- mice, we observed osteoclast-like giant cells, albeit in low numbers, with single or two nuclei, engulfing the mineralized extracellular matrix. TEM revealed that these giant cells contained large numbers of mitochondria, vesicles/vacuoles, and clear zone-like structures but no ruffled borders. They often engulfed fragmented bony/cartilaginous components of the extracellular matrix that had been degraded. Additionally, osteoclast-like giant cells exhibited immunoreactivity for vacuolar H+-ATPase, galectin-3, and siglec-15 but not for tartrate-resistant acid phosphatase, cathepsin K, or MMP-9, all of which are classical hallmarks of osteoclasts. Furthermore, osteoclast-like giant cells were ephrinB2-positive as they were near EphB4-positive osteoblasts that are also positive for alkaline phosphatase and Runx2 in Rankl-/- mice. Unlike Rankl-/- mice, c-fos-/- mice lacking osteoclast progenitors and mature osteoclasts had no ephrinB2-positive osteoclast-like cells or alkaline phosphatase-positive/Runx2-reactive osteoblasts. This suggests that similar to authentic osteoclasts, osteoclast-like giant cells might have the potential to activate osteoblasts in Rankl-/- mice. CONCLUSIONS: It seems plausible that osteoclast-like giant cells may have acquired some osteoclastic traits and the ability to resorb mineralized matrices even when the absence of RANK/RANKL signaling halted the osteoclastic differentiation cascade.

Histochemical assessment on osteoclasts in long bones of toll-like receptor 2 (TLR2) deficient mice.

OBJECTIVE: Toll-like receptor 2 (TLR2), recognizes a wide variety of pathogen-associated molecular patterns such as lipopolysaccharides, peptidoglycans, and lipopeptides, and is generally believed to be present in monocytes, macrophages, dendritic cells, and vascular endothelial cells. However, no histological examination of osteoclasts, which differentiate from precursors common to macrophages/monocytes, has been performed in a non-infected state of TLR2 deficiency. The objective of this study was to examine the histological properties and function of osteoclasts in the long bones of 8-week-old male TLR2 deficient (TLR2-/-) mice to gain insight into TLR2 function in biological circumstances without microbial infection. METHODS: Eight-week-old male wild-type and TLR2-/- mice were fixed with paraformaldehyde solution, and their tibiae and femora were used for micro-CT analysis, immunohistochemistry, transmission electron microscopy, and real-time PCR analysis. RESULTS: TLR2-/- tibiae and femora exhibited increased bone volume of metaphyseal trabeculae and elevated numbers of TRAP-positive osteoclasts. However, the number of multinucleated TRAP-positive osteoclasts was reduced, whereas mononuclear TRAP-positive cells increased, despite the high expression levels of Dc-Stamp and Oc-Stamp. Although TRAP-positive multinucleated and mononuclear osteoclasts showed the immunoreactivity and elevated expression of RANK and siglec-15, they revealed weak cathepsin K-positivity and less incorporation of the mineralized bone matrix, and often missing ruffled borders. It seemed likely that, despite the increased numbers, TLR2-/- osteoclasts reduced cell fusion and bone resorption activity. CONCLUSION: It seems likely that even without bacterial infection, TLR2 might participate in cell fusion and subsequent bone resorption of osteoclasts.

Evocalcet Rescues Secondary Hyperparathyroidism-driven Cortical Porosity in CKD Male Rats.

To elucidate the effect of evocalcet, a new oral calcimimetic to bone of secondary hyperparathyroidism (SHPT) with chronic kidney disease (CKD), the rats were 5/6 nephrectomized and fed on a high-phosphate diet. The treated rats were then divided into vehicle groups and evocalcet administered groups. The rats in the vehicle groups exhibited increased levels of serum PTH and inorganic phosphate (Pi) levels, high bone turnover, and severe cortical porosity, mimicking SHPT (CKD-SHPT rats). The cortical bone of the CKD-SHPT rats showed broad demineralization around the osteocytes, suppression of Phex/small integrin-binding ligand N-linked glycoprotein-mediated mineralization in the periphery of the osteocytic lacunae, and increased levels of osteocytic cell death, all of which were considered as the first steps of cortical porosity. In contrast, evocalcet ameliorated the increased serum PTH levels, the enlarged osteocytic lacunae, and the cortical porosity of the CKD-SHPT rats. Osteocytes of CKD-SHPT rats strongly expressed PTH receptor and Pit1/Pit2, which sense extracellular Pi, indicating that PTH and Pi affected these osteocytes. Cell death of cultured osteocytes increased in a Pi concentration-dependent manner, and PTH administration rapidly elevated Pit1 expression and enhanced osteocytic death, indicating the possibility that the highly concentrated serum PTH and Pi cause severe perilacunar osteolysis and osteocytic cell death. It is likely therefore that evocalcet not only decreases serum PTH but also reduces the exacerbation combined with PTH and Pi to the demineralization of osteocytic lacunae and osteocytic cell death, thereby protecting cortical porosity in CKD-SHPT rats.

Histochemical assessment of accelerated bone remodeling and reduced mineralization in Il-6 deficient mice.

OBJECTIVES: Interleukin-6 (IL-6) contributes to the regulation of functions in various tissues and organs. Even though IL-6 has been reported to modulate bone metabolism in previous studies, this finding is controversial. This study aims to evaluate the possible involvement of IL-6 in bone metabolism by examining the histological activity of osteoblasts and osteoclasts in the femora of Il-6 deficient (Il-6-/-) mice. METHODS: Eight-week-old male Il-6-/- mice and their wild-type littermates were fixed with a paraformaldehyde solution, and their femora were extracted for micro-CT analysis, immunohistochemistry, and real-time PCR analysis. RESULTS: Il-6-/- femora showed an increased bone volume/tissue volume (TV) but a reduced bone mineral density compared with the wild-type. Furthermore, the tissue-nonspecific alkaline phosphatase positive area/TV ratio, the expression of Runx2, Osterix, and Rankl, and the number of tartrate-resistant acid phosphatase-positive osteoclasts were all increased in the Il-6-/- mice. A considerable number of unmineralized areas within the bone matrix and abundant sclerostin-reactive osteocytes were observed in Il-6-/- femoral metaphyses but not in the wild-type. Interestingly, the gene expression of Cd206 was elevated in Il-6-/- femora, and many F4/80-positive macrophages/monocytes and CD206-immunoreactive macrophages in the primary trabeculae had migrated closer to the growth plate, where intense RANKL immunoreactivity was detected. These results suggest that, in an IL-6-deficient state, CD206-positive macrophages may differentiate into osteoclasts when in contact with RANKL-reactive osteoblastic cells. CONCLUSION: In a state of IL-6 deficiency, the population and cell activities of osteoblast, osteoclasts, and macrophages seemed to be facilitated, except for the reduced mineralization in bone.

Histochemical assessment on osteocytic osteolysis in lactating mice fed with a calcium-insufficient diet.

OBJECTIVES: This study aimed to examine if feeding lactating mice a calcium-insufficient diet while simultaneously administering alendronate (ALN) could potentially induce osteocytic osteolysis. METHODS: Lactating mice were fed calcium (Ca)-insufficient diets with or without ALN administration, and then their femurs were examined for TRAP and ALP, and observed by Kossa staining and transmission electron microscopy (TEM). Mice that had been fed a Ca-insufficient diet were then fed a 44Ca-containing diet, and their tibial sections were examined by isotope microscopy. RESULTS: Mice fed a Ca-insufficient diet had a reduced number of TRAP-positive osteoclasts after ALN administration. ALN-treated, lactating mice fed a Ca-insufficient diet had enlarged lacunae in their cortical bones, and TEM imaging demonstrated expanded regions between osteocytes and lacunar walls. In ALN-treated lactating mice fed a Ca-insufficient diet, huge areas of demineralized bone matrix occurred, centered around blood vessels in the cortical bone. Isotope microscopy showed 44Ca in the vicinity of the osteocytic lacunae, and in the broad, previously demineralized region around the blood vessels in the cortical bone of lactating mice fed a 44Ca-sufficient diet. CONCLUSIONS: Bone demineralization likely takes place in the periphery of the osteocytic lacunae and in the broad regions around the blood vessels of lactating mice when they are exposed to severely reduced serum Ca through a Ca-insufficient diet coupled with ALN administration.

Matrix Vesicle-Mediated Mineralization and Osteocytic Regulation of Bone Mineralization.

Bone mineralization entails two mineralization phases: primary and secondary mineralization. Primary mineralization is achieved when matrix vesicles are secreted by osteoblasts, and thereafter, bone mineral density gradually increases during secondary mineralization. Nearby extracellular phosphate ions (PO43-) flow into the vesicles via membrane transporters and enzymes located on the vesicles' membranes, while calcium ions (Ca2+), abundant in the tissue fluid, are also transported into the vesicles. The accumulation of Ca2+ and PO43- in the matrix vesicles induces crystal nucleation and growth. The calcium phosphate crystals grow radially within the vesicle, penetrate the vesicle's membrane, and continue to grow outside the vesicle, ultimately forming mineralized nodules. The mineralized nodules then attach to collagen fibrils, mineralizing them from the contact sites (i.e., collagen mineralization). Afterward, the bone mineral density gradually increases during the secondary mineralization process. The mechanisms of this phenomenon remain unclear, but osteocytes may play a key role; it is assumed that osteocytes enable the transport of Ca2+ and PO43- through the canaliculi of the osteocyte network, as well as regulate the mineralization of the surrounding bone matrix via the Phex/SIBLINGs axis. Thus, bone mineralization is biologically regulated by osteoblasts and osteocytes.

Histological functions of parathyroid hormone on bone formation and bone blood vessels.

BACKGROUND: The intermittent administration of parathyroid hormone (PTH) has been prescribed to osteoporotic patients due to its bone anabolic effects. In addition to its actions on bone cells, PTH appears to affect bone-specific blood vessels. These blood vessels are derived from bone marrow sinusoids, which express EphB4, a hallmark of veinous vascular endothelial cells. Given the presence of osteo-vascular interactions, it is important to elucidate the effects of PTH on bone cells and blood vessels in murine models. HIGHLIGHTS: PTH stimulates preosteoblastic proliferation and osteoblastic bone formation. The former appears to be directly affected by PTH, whereas the latter requires osteoclast-mediated coupling. The administration of PTH through high-frequency dosage schemes accelerates bone turnover featuring remodeling-based bone formation, whereas low-frequency schemes cause mainly remodeling-based and partly modeling-based bone formation. Normally, many blood vessels lack alpha smooth muscle actin (αSMA)-immunoreactive vascular muscle cells surrounding basement membranes, indicating them being capillaries. However, PTH administration increases the number of blood vessels surrounded by αSMA-positive cells. These αSMA-positive cells spread out of blood vessels and express alkaline phosphatase and c-kit, suggesting their potential to differentiate into osteogenic and vascular endothelial/perivascular cells. Unlike bone cells, αSMA-positive cells did not appear in the periphery of blood vessels in the kidney and liver, and the thickness of the tunica media did not change regardless of PTH administration. CONCLUSION: Based on the results of the study and presence of osseous-vascular interactions, PTH appears to influence not only osteoblastic cells, but also blood vessels in bone.

Histochemical examination of blood vessels in murine femora with intermittent PTH administration.

OBJECTIVE: To verify the biological effects of parathyroid hormone (PTH) on the blood vessels in the bone, this study aimed to investigate histological alterations in endomucin-positive blood vessels and perivascular cells in murine femora after intermittent PTH administration. For comparison with blood vessels in the bone, we examined the distribution of endomucin-positive blood vessels and surrounding αSMA-immunoreactive perivascular cells in the liver, kidney, and aorta with or without PTH administration. METHODS: Six-week-old male C57BL/6J mice received hPTH [1-34] or vehicle for two weeks. All mice were fixed with a paraformaldehyde solution after euthanasia, and the right femora, kidney, liver, and aorta were extracted for immunohistochemical analysis of endomucin, αSMA, ephrinB2, EphB4, and HIF1α. Light microscopic observations of semi-thin sections and transmission electron microscopic (TEM) observations of ultra-thin sections were performed on the left femora. RESULTS: After intermittent PTH administration, αSMA-reactive/ephrinB2-positive stromal cells appeared around endomucin-positive/EphB4-immunoreactive blood vessels in the bone. In addition, intense immunoreactivities of EphB4 and HIF1α were seen in vascular endothelial cells after the PTH treatment. Several stromal cells surrounding PTH-treated blood vessels exhibited well-developed rough endoplasmic reticulum under TEM observations. In contrast to bone tissues, αSMA-positive stromal cells did not increase around the endomucin-positive blood vessels in the kidney, liver, or aorta, even after PTH administration. CONCLUSION: These findings show that intermittent PTH administration increases αSMA-reactive/ephrinB2-positive perivascular stromal cells in bone tissue but not in the kidney, liver, or aorta, suggesting that PTH preferentially affects blood vessels in the bone.

Immunolocalization of endomucin-reactive blood vessels and α-smooth muscle actin-positive cells in murine nasal conchae.

OBJECTIVES: Recently, the biological functions of endomucin-positive blood vessels and closely associated αSMA-positive cells in long bones have been highlighted. The surrounding tissues of the flat bones, such as nasal bones covered with mucosa and lamina propria, are different from those of the long bones, indicating the different distributions of endomucin-positive blood vessels and αSMA-reactive cells in nasal bones. This study demonstrates the immunolocalization of endomucin-reactive blood vessels and αSMA-positive cells in the nasal conchae of 3- and 7-week-old mice. METHODS: The nasal conchae of 3-week-old and 7-week-old male C57BL/6J mice were used for immunoreaction of endomucin, CD34, PDGFbb, TRAP, and c-kit. RESULTS: While we identified abundant endomucin-reactive blood vessels in the lamina propria neighboring the bone, not all were positive for endomucin. More CD34-reactive cells and small blood vessels were observed in the nasal conchae of 3-week-old mice than in those of 7-week-old mice. Some αSMA-positive cells in the nasal conchae surrounded the blood vessels, indicating vascular smooth muscle cells, while other αSMA-immunopositive fibroblastic cells were detected throughout the lamina propria. αSMA-positive cells did not co-localize with c-kit-immunoreactivity, thereby indicating that the αSMA-positive cells may be myofibroblasts rather than undifferentiated mesenchymal cells. CONCLUSIONS: Unlike long bones, nasal conchae contain endomucin-positive as well as endomucin-negative blood vessels and exhibit numerous αSMA-positive fibroblastic cells throughout the lamina propria neighboring the bone. Apparently, the distribution patterns of endomucin-positive blood vessels and αSMA-positive cells in nasal conchae are different from those in long bones.

Bone biopsy findings in patients receiving long-term bisphosphonate therapy for glucocorticoid-induced osteoporosis.

INTRODUCTION: Bisphosphonates (BPs) have been shown to reduce the incidence of vertebral fractures during the first year or two of glucocorticoid (GC) treatments and are therefore recommended as a first-line treatment for GC-induced osteoporosis (GIO). However, there are theoretical concerns about the long-term use of BPs in low-turnover osteoporosis caused by chronic GC therapy. MATERIALS AND METHODS: We analyzed the trabecular microarchitecture, bone metabolism, and material strength of iliac crest bone biopsy samples from 10 female patients with rheumatoid arthritis who received an average of 6.7 years of BP therapy for GIO (GIOBP group), compared with those of 10 age- and bone mineral density (BMD)-matched non-rheumatoid arthritis postmenopausal women (reference group). RESULTS: Patients in the GIOBP group had a significantly greater fracture severity index, as calculated from the number and the extent of vertebral fractures compared with the reference patients. Micro-computed tomography analysis showed that the degree of mineralization and trabecular microarchitecture were significantly lower in the GIOBP group than in the reference patients. Patients in the GIOBP group exhibited lower bone contact stiffness, determined by micro-indentation testing, than in the reference group. The contact stiffness of the bone was negatively correlated with the fracture severity index and the daily prednisolone dosage. Immunohistochemistry and serum bone turnover markers showed decreased osteoclastic activity, impaired mineralization, and an increased fraction of empty lacunae in the GIOBP group. CONCLUSION: Our findings indicate that patients receiving long-term BP for GIO are still at high risk for fragility fractures because of poor bone quality.

Characterization, pharmacokinetics, and pharmacodynamics of anti-Siglec-15 antibody and its potency for treating osteoporosis and as follow-up treatment after parathyroid hormone use.

Recent studies have established the idea that Siglec-15 is involved in osteoclast differentiation and/or function, and it is anticipated that therapies suppressing Siglec-15 function can be used to treat bone diseases such as osteoporosis. We have produced rat monoclonal anti-Siglec-15 antibody (32A1) and successively generated humanized monoclonal anti-Siglec-15 antibody (DS-1501a) from 32A1. Studies on the biological properties of DS-1501a showed its specific binding affinity to Siglec-15 and strong activity to inhibit osteoclastogenesis. 32A1 inhibited multinucleation of osteoclasts and bone resorption (pit formation) in cultured mouse bone marrow cells. 32A1 also inhibited pit formation in cultured human osteoclast precursor cells. Maximum serum concentration and serum exposure of DS-1501a in rats were increased in a dose-dependent manner after single subcutaneous or intravenous administration. Furthermore, single administration of DS-1501a significantly suppressed bone resorption markers with minimal effects on bone formation markers and suppressed the decrease in bone mineral density (BMD) of the lumbar vertebrae in ovariectomized (OVX) rats. In histological analysis, the osteoclasts distant from the chondro-osseous junction of the tibia tended to be flattened, shrunken, and functionally impaired in 32A1-treated rats, while alkaline phosphatase-positive osteoblasts were observed throughout the metaphyseal trabeculae. In addition, we compared the efficacy of 32A1 with that of alendronate (ALN) as follow-up medicine after treatment with parathyroid hormone (PTH) using mature established osteoporosis rats. The beneficial effect of PTH on bone turnover disappeared 8 weeks after discontinuing the treatment. The administration of 32A1 once every 4 weeks for 8 weeks suppressed bone resorption and bone formation when the treatment was switched from PTH to 32A1, leading to the maintenance of BMD and bone strength. Unlike with ALN, the onset of suppression of bone resorption with 32A1 was rapid, while the suppression of bone formation was mild. The improvement of bone mass, beneficial bone turnover balance, and suppression of osteoclast differentiation/multinucleation achieved by 32A1 were supported by histomorphometry. Notably, the effects of 32A1 on bone strength, not only structural (extrinsic) but also material (intrinsic) properties, were significantly greater than those of ALN. Since the effect of 32A1 on BMD was moderate, its effect on bone strength could not be fully explained by the increase in BMD. The beneficial balance of bone turnover caused by 32A1 might, at least in part, be responsible for the improvement in bone quality. This is the first report describing the effects of anti-Siglec-15 antibody in OVX rats; the findings suggest that this antibody could be an excellent candidate for treating osteoporosis, especially in continuation therapy after PTH treatment, due to its rapid action and unprecedented beneficial effects on bone quality.