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Background Zinc‐finger E‐box‐binding homeobox‐1 (ZEB1) is predominantly found in type‐H vessels. However, the roles of ZEB1 and type‐H vessels in steroid‐induced osteonecrosis of the femoral head (SONFH) are unclear. Methods Human femoral heads were collected to detect the expression of ZEB1 and the levels of type‐H vessels. Then, the SONFH model was developed by injecting C57BL/6 mice with lipopolysaccharide and methylprednisolone. Micro‐computed tomography, angiography, double calcein labeling, immunofluorescence, immunohistochemistry, quantitative real‐time polymerase chain reaction, and Western blotting were performed to detect the expression of ZEB1, the Wnt/β‐catenin pathway, type‐H vessels, and the extent to which ZEB1 mediates angiogenesis and osteogenesis. Human umbilical vein endothelial cells were also used to explore the relationship between ZEB1 and the Wnt/β‐catenin pathway. Results We found that ZEB1 expression and the formation of type‐H vessels decreased in SONFH patients and in a mouse model. The number of vascular endothelial growth factors in the femoral heads also decreased. Moreover, the bone mineral density, trabecular number, mineral apposition rate, and expression of genes related to osteogenesis decreased. After ZEB1 knockdown, angiogenesis and osteogenesis decreased. However, the numbers of type‐H vessels and the extent of angiogenesis and osteogenesis improved after activation of the Wnt/β‐catenin pathway. Conclusions The ZEB1 expression decreased in SONFH, causing a decrease in type‐H vessel, and it mediated angiogenesis and osteogenesis by regulating the Wnt/β‐catenin pathway, ultimately accelerating the process of SONFH. The application of glucocorticoid could restrain the expression of ZEB1, causing a decrease in the expression of β‐catenin, transcription factor‐4, C‐Myc, and other key genes in the Wnt/β‐catenin pathway, ultimately damaging the formation of type‐H vessels and aggravating necrosis of the femoral head. However, activation of the Wnt/β‐catenin pathway promotes the formation of type‐H vessels and prevents SONFH (steroid‐induced osteonecrosis of the femoral head).

Improving revascularization is one of the major measures in fracture treatment. Moderate local inflammation triggers angiogenesis, whereas systemic inflammation hampers angiogenesis. Previous studies showed that Akkermansia muciniphila, a gut probiotic, ameliorates systemic inflammation by tightening the intestinal barrier. In this study, fractured mice intragastrically administrated with A. muciniphila were found to display better fracture healing than mice treated with vehicle. Notably, more preosteclasts positive for platelet-derived growth factor-BB (PDGF-BB) were induced by A. muciniphila at 2 weeks post fracture, coinciding with increased formation of type H vessels, a specific vessel subtype that couples angiogenesis and osteogenesis, and can be stimulated by PDGF-BB. Moreover, A. muciniphila treatment significantly reduced gut permeability and inflammation at the early stage. Dextran sulfate sodium (DSS) was used to disrupt the gut barrier to determine its role in fracture healing and whether A. muciniphila still can stimulate bone fracture healing. As expected, A. muciniphila evidently improved gut barrier, reduced inflammation and restored the impaired bone healing and angiogenesis in DSS-treated mice. Our results suggest that A. muciniphila reduces intestinal permeability and alleviates inflammation, which probably induces more PDGF-BB+ preosteoclasts and type H vessel formation in callus, thereby promoting fracture healing. This study provides the evidence for the involvement of type H vessels in fracture healing and suggests the potential of A. muciniphila as a promising strategy for bone healing. This article has an associated First Person interview with the first author of the paper.

Macrophages are essential players in the process of fracture healing, acting by remodeling of the extracellular matrix and enabling vascularization. Whilst activated macrophages of M1-like phenotype are present in the initial pro-inflammatory phase of hours to days of fracture healing, an anti-inflammatory M2-like macrophage phenotype is supposed to be crucial for the induction of downstream cascades of healing, especially the initiation of vascularization. In a mouse-osteotomy model, we provide a comprehensive characterization of vessel (CD31 , Emcn ) and macrophage phenotypes (F4/80, CD206, CD80, Mac-2) during the process of fracture healing. To this end, we phenotype the phases of vascular regeneration-the expansion phase (d1-d7 after injury) and the remodeling phase of the endothelial network, until tissue integrity is restored (d14-d21 after injury). Vessels which appear during the bone formation process resemble type H endothelium (CD31 Emcn ), and are closely connected to osteoprogenitors (Runx2 , Osx ) and F4/80 macrophages. M1-like macrophages are present in the initial phase of vascularization until day 3 post osteotomy, but they are rare during later regeneration phases. M2-like macrophages localize mainly extramedullary, and CD206 macrophages are found to express Mac-2 during the expansion phase. VEGFA expression is initiated by CD80 cells, including F4/80 macrophages, until day 3, while subsequently osteoblasts and chondrocytes are main contributors to VEGFA production at the fracture site. Using Longitudinal Intravital Microendoscopy of the Bone (LIMB) we observe changes in the motility and organization of CX3CR1 cells, which infiltrate the injury site after an osteotomy. A transient accumulation, resulting in spatial polarization of both, endothelial cells and macrophages, in regions distal to the fracture site, is evident. Immunofluorescence histology followed by histocytometric analysis reveals that F4/80 CX3CR1 myeloid cells precede vascularization.

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a key enzyme that transform cortisone to cortisol, which activates the endogenous glucocorticoid function. 11β-HSD1 has been observed to regulate skeletal metabolism, specifically within osteoblasts. However, the function of 11β-HSD1 in osteoclasts has not been elucidated. In this study, we observed increased 11β-HSD1 expression in osteoclasts within an osteoporotic mice model (ovariectomized mice). Then, 11β-HSD1 global knock-out or knock-in mice were employed to demonstrate its function in manipulating bone metabolism, showing significant bone volume decrease in 11β-HSD1 knock-in mice. Furthermore, specifically knock out 11β-HSD1 in osteoclasts, by crossing cathepsin-cre mice with 11β-HSD1 mice, presented significant protecting effect of skeleton when they underwent ovariectomy surgery. experiments showed the endogenous high expression of 11β-HSD1 lead to osteoclast formation and maturation. Meanwhile, we found 11β-HSD1 facilitated mature osteoclasts formation inhibited bone formation coupled H type vessel (CD31 Emcn ) growth through reduction of PDFG-BB secretion. Finally, transcriptome sequencing of 11β-HSD1 knock in osteoclast progenitor cells indicated the Hippo pathway1 was mostly enriched. Then, by suppression of YAP expression in Hippo signaling, we observed the redundant of osteoclasts formation even in 11β-HSD1 high expression conditions. In conclusion, our study demonstrated the role of 11β-HSD1 in facilitating osteoclasts formation and maturation through the Hippo signaling, which is a new therapeutic target to manage osteoporosis.

Background Osteoporosis, resulting from an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation, affects millions globally. Recent studies have identified type H vessels (CD31 hi EMCN hi ) as a specialized subset of bone blood vessels that positively regulate bone formation. This study aims to investigate the effects of metformin on bone mass, strength, and angiogenesis in osteoporotic mice, and to elucidate the underlying molecular mechanisms, particularly focusing on the YAP1/TAZ-HIF1α axis. Methods Osteoporotic mice were administered metformin, and bone mass and strength were measured. In vivo and in vitro angiogenesis assays were performed under hypoxic conditions. Expression levels of YAP1/TAZ and HIF1α were assessed in femoral metaphysis and hypoxia-cultured human microvascular endothelial cells (HMECs). Small interfering RNA was used to interfere with HIF1α or YAP1/TAZ expression in hypoxia-cultured HMECs. Additionally, we employed AAV-mediated overexpression of YAP1/TAZ in vivo to determine whether elevated YAP1/TAZ levels alter metformin’s effects on bone mass and angiogenesis. Results Metformin significantly enhanced bone mass and strength in osteoporotic mice. It also promoted angiogenesis under hypoxia conditions both in vivo and in vitro. Metformin reduced YAP1/TAZ expression while increasing HIF1α expression in both the femoral metaphysis of osteoporotic mice and hypoxia-cultured HMECs. Interference with HIF1α or YAP1/TAZ confirmed that metformin enhances HIF1α and its target genes primarily by inhibiting YAP1/TAZ. Furthermore, overexpression of YAP1/TAZ partially reversed the bone-protective effect of metformin, leading to reduced HIF1α levels and diminished type H vessel formation. Conclusion Our findings suggest that metformin holds promise as a therapeutic agent for osteoporosis by enhancing type H vessel formation through the inhibition of the YAP1/TAZ-HIF1α axis. Graphical abstract

Background Bone tissue engineering is a new concept bringing hope for the repair of large bone defects, which remains a major clinical challenge. The formation of vascularized bone is key for bone tissue engineering. Growth of specialized blood vessels termed type H is associated with bone formation. In vivo and in vitro studies have shown that low level laser therapy (LLLT) promotes angiogenesis, fracture healing, and osteogenic differentiation of stem cells by increasing reactive oxygen species (ROS). However, whether LLLT can couple angiogenesis and osteogenesis, and the underlying mechanisms during bone formation, remains largely unknown. Methods Mouse bone marrow mesenchymal stem cells (BMSCs) combined with biphasic calcium phosphate (BCP) grafts were implanted into C57BL/6 mice to evaluate the effects of LLLT on the specialized vessel subtypes and bone regeneration in vivo. Furthermore, human BMSCs and human umbilical vein endothelial cells (HUVECs) were co-cultured in vitro. The effects of LLLT on cell proliferation, angiogenesis, and osteogenesis were assessed. Results LLLT promoted the formation of blood vessels, collagen fibers, and bone tissue and also increased CD31 hi EMCN hi -expressing type H vessels in mBMSC/BCP grafts implanted in mice. LLLT significantly increased both osteogenesis and angiogenesis, as well as related gene expression (HIF-1α, VEGF, TGF-β) of grafts in vivo and of co-cultured BMSCs/HUVECs in vitro. An increase or decrease of ROS induced by H 2 O 2 or Vitamin C, respectively, resulted in an increase or decrease of HIF-1α, and a subsequent increase and decrease of VEGF and TGF-β in the co-culture system. The ROS accumulation induced by LLLT in the co-culture system was significantly decreased when HIF-1α was inhibited with DMBPA and was followed by decreased expression of VEGF and TGF-β. Conclusions LLLT enhanced vascularized bone regeneration by coupling angiogenesis and osteogenesis. ROS/HIF-1α was necessary for these effects of LLLT. LLLT triggered a ROS-dependent increase of HIF-1α, VEGF, and TGF-β and resulted in subsequent formation of type H vessels and osteogenic differentiation of mesenchymal stem cells. As ROS also was a target of HIF-1α, there may be a positive feedback loop between ROS and HIF-1α, which further amplified HIF-1α induction via the LLLT-mediated ROS increase. This study provided new insight into the effects of LLLT on vascularization and bone regeneration in bone tissue engineering.

Blood vessels are essential for bone development and metabolism. Type H vessels in bone, named after their high expression of CD31 and Endomucin (Emcn), have recently been reported to locate mainly in the metaphysis, exhibit different molecular properties and couple osteogenesis and angiogenesis. A strong correlation between type H vessels and bone metabolism is now well‐recognized. The crosstalk between type H vessels and osteoprogenitor cells is also involved in bone metabolism‐related diseases such as osteoporosis, osteoarthritis, fracture healing and bone defects. Targeting the type H vessel formation may become a new approach for managing a variety of bone diseases. This review highlighted the roles of type H vessels in bone‐related diseases and summarized the research attempts to develop targeted intervention, which will help us gain a better understanding of their potential value in clinical application.

Ginsenoside Rg1 (Rg1) has been demonstrated to have antidiabetic and antiosteoporotic activities. The aim of this study was to investigate the protective effect of Rg1 against diabetic osteoporosis and the underlying mechanism. In vitro , we found that Rg1 increased the number of osteoprogenitors and alleviated high glucose (HG) induced apoptosis of osteoprogenitors by MTT assays and flow cytometry. qRT‒PCR and western blot analysis suggested that Rg1 can also promote the secretion of vascular endothelial growth factor (VEGF) by osteoprogenitors and promote the coupling of osteogenesis and angiogenesis. Rg1 can also promote the proliferation of human umbilical vein endothelial cells (HUVECs) cultured in high glucose, enhance the angiogenic ability of endothelial cells, and activate the Notch pathway to promote endothelial cells to secrete the osteogenesis-related factor Noggin to regulate osteogenesis, providing further feedback coupling of angiogenesis and osteogenesis. Therefore, we speculated that Rg1 may have similar effects on type H vessels. We used the Goto-Kakizaki (GK) rat model to perform immunofluorescence staining analysis on two markers of type H vessels, Endomucin (Emcn) and CD31, and the osteoblast-specific transcription factor Osterix, and found that Rg1 stimulates type H angiogenesis and bone formation. In vivo experiments also demonstrated that Rg1 promotes VEGF secretion, activates the Noggin/Notch pathway, increases the level of coupling between type H vessels and osteogenesis, and improves the bone structure of GK rats. All of these data reveal that Rg1 is a promising candidate drug for treating diabetic osteoporosis as a potentially bioactive molecule that promotes angiogenesis and osteointegration coupling.

Type 1 diabetes mellitus (T1DM) can lead to severe diabetic osteopathy, largely driven by alterations in the bone marrow hypoxic microenvironment and damage to type H vessels. This study employed multimodal imaging—including DCE-MRI, Micro-CT, and USPIO-enhanced MRI—to enable early in vivo assessment of hypoxic changes and type H vessel impairment in a T1DM rabbit model. Experiments involved 20 rabbits with alloxan-induced T1DM and controls, evaluated four months post-modeling. Imaging revealed significant differences in bone marrow microcirculatory perfusion and vascular permeability in T1DM rabbits, along with elevated USPIO uptake and regional heterogeneity that correlated with type H vessel distribution. Accompanying pathological changes were confirmed via immunofluorescence, qPCR, and transmission electron microscopy, suggesting an association of the AGEs/ROS-HIF-1α-VEGF pathway with these microvascular lesions. Our findings offer visual and quantitative imaging evidence to inform future clinical strategies targeting type H vessel hypoxia in diabetic osteopathy.

The challenges posed by delayed atrophic healing and nonunion stand as formidable obstacles in osteoporotic fracture treatment. The processes of type H angiogenesis and osteogenesis emerge as pivotal mechanisms during bone regeneration. Notably, the preconditioning of adipose-derived stem cell (ADSC) exosomes under hypoxic conditions has garnered attention for its potential to augment the secretion and functionality of these exosomes. In the present investigation, we embarked upon a comprehensive elucidation of the underlying mechanisms of hypo-ADSC-Exos within the milieu of osteoporotic bone regeneration. Our findings revealed that hypo-ADSC-Exos harboured a preeminent miRNA, namely, miR-21-5p, which emerged as the principal orchestrator of angiogenic effects. Through in vitro experiments, we demonstrated the capacity of hypo-ADSC-Exos to stimulate the proliferation, migration, and angiogenic potential of human umbilical vein endothelial cells (HUVECs) via the mediation of miR-21-5p. The inhibition of miR-21-5p effectively attenuated the proangiogenic effects mediated by hypo-ADSC-Exos. Mechanistically, our investigation revealed that exosomal miR-21-5p emanating from hypo-ADSCs exerts its regulatory influence by targeting sprouly1 (SPRY1) within HUVECs, thereby facilitating the activation of the PI3K/AKT signalling pathway. Notably, knockdown of SPRY1 in HUVECs was found to potentiate PI3K/AKT activation and, concomitantly, HUVEC proliferation, migration, and angiogenesis. The culminating stage of our study involved a compelling in vivo demonstration wherein GelMA loaded with hypo-ADSC-Exos was validated to substantially enhance local type H angiogenesis and concomitant bone regeneration. This enhancement was unequivocally attributed to the exosomal modulation of SPRY1. In summary, our investigation offers a pioneering perspective on the potential utility of hypo-ADSC-Exos as readily available for osteoporotic fracture treatment. Graphical Abstract