BACKGROUND:Diabetic osteoporosis (DOP), a severe complication of diabetes mellitus, arises primarily from hyperglycemia-induced chronic inflammation and remains challenging to treat. Corylin, a flavonoid isolated from Psoralea corylifolia l., has been reported to possess anti-inflammatory properties; however, its therapeutic potential for DOP remains unknown. PURPOSE:This study aimed to determine the therapeutic potential of corylin on DOP and to elucidate its underlying mechanisms preliminarily. METHODS:The effects of corylin on lipopolysaccharide-induced inflammatory responses in macrophages, as well as its impacts on the anti-osteoblastic and pro-osteoclastic abilities of activated macrophages, were assessed by quantitative real-time PCR analysis and cytochemical staining. In vivo, the effects of corylin in a streptozotocin-induced DOP mouse model were evaluated using microcomputed tomography, biomechanical testing, immunohistochemistry, and immunofluorescence. Molecular docking analysis was employed to identify potential targets of corylin. Surface plasmon resonance experiments were performed to validate the docking-predicted corylin-target interactions and to quantify binding affinity and kinetics. Subsequently, the roles of the potential target in the regulatory effects of corylin on osteogenesis, osteoclastogenesis, and inflammation were evaluated by using small interfering RNA in bone marrow mesenchymal stem cells, bone marrow macrophages, and RAW264.7 cells, as well as by using recombinant adeno-associated virus in mice. RESULTS:In vitro, corylin not only enhanced osteoblast differentiation and suppressed osteoclast formation, but also reduced the secretion of pro-inflammatory cytokines in lipopolysaccharide-activated macrophages, thereby impairing their anti-osteoblastic and pro-osteoclastic activities. Consistently, in vivo studies demonstrated that intragastric administration of corylin significantly improved bone mass and microarchitecture in streptozotocin-induced DOP mice by promoting bone formation, inhibiting bone resorption, and alleviating local and systemic inflammation. Mechanistically, these beneficial effects depended on recombination-activating gene 1 (RAG1), as RAG1 knockdown abolished corylin's effects on bone metabolism and inflammation, both in vitro and in vivo. CONCLUSION:This study provides the first evidence of corylin's efficacy in a diabetic osteoporosis model and identifies RAG1 as its direct binding target, highlighting a previously unrecognized role of RAG1 in regulating bone metabolism and inflammation.

Skin injury refers to the disruption of skin integrity, resulting in the loss of normal tissue, which can occur due to wounds, diseases, or surgeries. The repair of skin injuries is a highly regulated process involving cell migration, proliferation, collagen matrix deposition, and tissue remodeling, all of which are intricately linked to inflammation and angiogenesis. Angiogenesis—the formation of new blood vessels—plays a pivotal role in this process by ensuring the delivery of oxygen and nutrients that are essential for healing. Key angiogenic factors, such as vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor, are crucial in regulating vascular formation. Recent advances have shed light on the complex interactions between angiogenesis and inflammatory responses, emphasizing their critical roles in wound healing, scar formation, and tissue remodeling. However, effectively regulating angiogenesis in clinical settings remains challenging due to the complexity of its underlying mechanisms. This review offers a comprehensive analysis of angiogenesis in skin injury repair, focusing on its regulatory mechanisms, functional roles during different stages of healing, and therapeutic strategies aimed at optimizing vascularization for enhanced clinical outcomes. By advancing our understanding of angiogenesis, we seek to contribute to the development of innovative treatments for skin injuries.

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 (CD31hiEMCNhi) 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. 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. 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. 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.

Background Bone marrow mesenchymal stem cells (BMSCs) can undergo inadequate osteogenesis or excessive adipogenesis as they age due to changes in the bone microenvironment, ultimately resulting in decreased bone density and elevated risk of fractures in senile osteoporosis. This study aims to investigate the effects of osteocyte senescence on the bone microenvironment and its influence on BMSCs during aging.Results Primary osteocytes were isolated from 2-month-old and 16-month-old mice to obtain young osteocyte-derived extracellular vesicles (YO-EVs) and senescent osteocyte-derived EVs (SO-EVs), respectively. YO-EVs were found to significantly increase alkaline phosphatase activity, mineralization deposition, and the expression of osteogenesis-related genes in BMSCs, while SO-EVs promoted BMSC adipogenesis. Neither YO-EVs nor SO-EVs exerted an effect on the osteoclastogenesis of primary macrophages/monocytes. Our constructed transgenic mice, designed to trace osteocyte-derived EV distribution, revealed abundant osteocyte-derived EVs embedded in the bone matrix. Moreover, mature osteoclasts were found to release osteocyte-derived EVs from bone slices, playing a pivotal role in regulating the functions of the surrounding culture medium. Following intravenous injection into young and elderly mouse models, YO-EVs demonstrated a significant enhancement of bone mass and biomechanical strength compared to SO-EVs. Immunostaining of bone sections revealed that YO-EV treatment augmented the number of osteoblasts on the bone surface, while SO-EV treatment promoted adipocyte formation in the bone marrow. Proteomics analysis of YO-EVs and SO-EVs showed that tropomyosin-1 (TPM1) was enriched in YO-EVs, which increased the matrix stiffness of BMSCs, consequently promoting osteogenesis. Specifically, the siRNA-mediated depletion of Tpm1 eliminated pro-osteogenic activity of YO-EVs both in vitro and in vivo.Conclusions Our findings suggested that YO-EVs played a crucial role in maintaining the balance between bone resorption and formation, and their pro-osteogenic activity declining with aging. Therefore, YO-EVs and the delivered TPM1 hold potential as therapeutic targets for senile osteoporosis.

Due to increasing morbidity worldwide, fractures are becoming an emerging public health concern. This study aimed to investigate the effect of metformin on the healing of osteoporotic as well as normal fractures. Type H vessels have recently been identified as a bone-specific vascular subtype that supports osteogenesis. Here, we show that metformin accelerated fracture healing in both osteoporotic and normal mice. Moreover, metformin promoted angiogenesis in vitro under hypoxia as well as type H vessel formation throughout fracture healing. Mechanistically, metformin increased the expression of HIF-1α, an important positive regulator of type H vessel formation, by inhibiting the expression of YAP1/TAZ in calluses and hypoxia-cultured human microvascular endothelial cells (HMECs). The results of HIF-1α or YAP1/TAZ interference in hypoxia-cultured HMECs using siRNA further suggested that the enhancement of HIF-1α and its target genes by metformin is primarily through YAP1/TAZ inhibition. Finally, overexpression of YAP1/TAZ partially counteracted the effect of metformin in promoting type H vessel-induced angiogenesis-osteogenesis coupling during fracture repair. In summary, our findings suggest that metformin has the potential to be a therapeutic agent for fractures by promoting type H vessel formation through YAP1/TAZ inhibition.

Obesity has recently been defined as a chronic low-grade inflammatory disease. Obesity-induced inflammation of adipose tissue (AT) is an essential trigger for insulin resistance (IR) and related metabolic diseases. Although the underlying molecular basis of this inflammation has not been fully identified, there is consensus that the recruited and activated macrophages in AT are the most important culprits of AT chronic inflammation. Adipose tissue macrophages (ATMs) are highly plastic and could be polarized from an anti-inflammatory M2 to proinflammatory M1 phenotypes on stimulation by microenvironmental signals from obese AT. Many efforts have been made to elucidate the molecular signaling pathways of macrophage polarization; however, the upstream drivers governing and activating macrophage polarization have rarely been summarized, particularly regulatory messages from the AT microenvironment. In addition to adipocytes, the AT bed also contains a variety of immune cells, stem cells, as well as vascular, neural, and lymphatic tissues throughout, which together orchestrate the AT microenvironment. Here, we summarize how the aforesaid neighbors of ATMs in the AT microenvironment send messages to ATMs and thus regulate its phenotype during obesity. Deciphering the biology and polarization of ATMs in the obese environment is expected to provide a precise immunotherapy for adipose inflammation and obesity-related metabolic diseases.

Adipocyte differentiation of bone marrow mesenchymal stem/stromal cells (BMSCs) instead of osteoblast formation contributes to age- and menopause-related marrow adiposity and osteoporosis. Vascular calcification often occurs with osteoporosis, a contradictory association called “calcification paradox”. Here we show that extracellular vesicles derived from aged bone matrix (AB-EVs) during bone resorption favor BMSC adipogenesis rather than osteogenesis and augment calcification of vascular smooth muscle cells. Intravenous or intramedullary injection of AB-EVs promotes bone-fat imbalance and exacerbates Vitamin D3 (VD3)-induced vascular calcification in young or old mice. Alendronate (ALE), a bone resorption inhibitor, down-regulates AB-EVs release and attenuates aging- and ovariectomy-induced bone-fat imbalance. In the VD3-treated aged mice, ALE suppresses the ovariectomy-induced aggravation of vascular calcification. MiR-483-5p and miR-2861 are enriched in AB-EVs and essential for the AB-EVs-induced bone-fat imbalance and exacerbation of vascular calcification. Our study uncovers the role of AB-EVs as a messenger for calcification paradox by transferring miR-483-5p and miR-2861.

Recently, the gut microbiota (GM) has been shown to be a regulator of bone homeostasis and the mechanisms by which GM modulates bone mass are still being investigated. Here, it is found that colonization with GM from children (CGM) but not from the elderly (EGM) prevents decreases in bone mass and bone strength in conventionally raised, ovariectomy (OVX)-induced osteoporotic mice. 16S rRNA gene sequencing reveals that CGM reverses the OVX-induced reduction of Akkermansia muciniphila (Akk). Direct replenishment of Akk is sufficient to correct the OVX-induced imbalanced bone metabolism and protect against osteoporosis. Mechanistic studies show that the secretion of extracellular vesicles (EVs) is required for the CGM- and Akk-induced bone protective effects and these nanovesicles can enter and accumulate into bone tissues to attenuate the OVX-induced osteoporotic phenotypes by augmenting osteogenic activity and inhibiting osteoclast formation. The study identifies that gut bacterium Akk mediates the CGM-induced anti-osteoporotic effects and presents a novel mechanism underlying the exchange of signals between GM and host bone.

Remodeling of energy-storing white fat into energy-consuming beige fat has led to a promising new approach to alleviate adiposity. Several studies have shown adipokines can induce white adipose tissue (WAT) beiging through autocrine or paracrine actions. Betatrophin, a novel adipokine, has been linked to energy expenditure and lipolysis but not clearly clarified. Here, we using high-fat diet-induced obesity to determine how betatrophin modulate beiging and adiposity. We found that betatrophin-knockdown mice displayed less white fat mass and decreased plasma TG and NEFA levels. Consistently, inhibition of betatrophin leads to the phenotype change of adipocytes characterized by increased mitochondria contents, beige adipocytes and mitochondria biogenesis-specific markers both in vivo and in vitro. Of note, blocking AMP-activated protein kinase (AMPK) signaling pathway is able to abolish enhanced beige-like characteristics in betatrophin-knockdown adipocytes. Collectively, downregulation of betatrophin induces beiging in white adipocytes through activation of AMPK signaling pathway. These processes suggest betatrophin as a latent therapeutic target for obesity.

Osteoporosis and osteoporotic fractures severely compromise quality of life in elderly people and lead to early death. Human umbilical cord mesenchymal stromal cell (MSC)-derived extracellular vesicles (hucMSC-EVs) possess considerable therapeutic effects in tissue repair and regeneration. Thus, in the present study, we investigated the effects of hucMSC-EVs on primary and secondary osteoporosis and explored the underlying mechanisms. Methods: hucMSCs were isolated and cultured. EVs were obtained from the conditioned medium of hucMSCs and determined by using transmission electron microscopy, dynamic light scattering and Western Blot analyses. The effects of hucMSC-EVs on ovariectomy-induced postmenopausal osteoporosis and tail suspension-induced hindlimb disuse osteoporosis in mouse models were assessed by using microcomputed tomography, biomechanical, histochemical and immunohistochemical, as well as histomorphometric analyses. Proteomic analysis was applied between hucMSC-EVs and hucMSCs to screen the candidate proteins that mediate hucMSC-EVs function. The effects of hucMSC-EVs on osteogenic and adipogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs), and osteoclastogenesis of the macrophage cell line RAW264.7 in vitro were determined by using cytochemical staining and quantitative real-time PCR analysis. Subsequently, the roles of the key protein in hucMSC-EVs-induced regulation on BMSCs and RAW264.7 cells were evaluated. Results: hucMSCs were able to differentiate into osteoblasts, adipocytes or chondrocytes and positively expressed CD29, CD44, CD73 and CD90, but negatively expressed CD34 and CD45. The morphological assessment revealed the typical cup- or sphere-shaped morphology of hucMSC-EVs with diameters predominantly ranging from 60 nm to 150 nm and expressed CD9, CD63, CD81 and TSG101. The systemic administration of hucMSC-EVs prevented bone loss and maintained bone strength in osteoporotic mice by enhancing bone formation, reducing marrow fat accumulation and decreasing bone resorption. Proteomic analysis showed that the potently pro-osteogenic protein, CLEC11A (C-type lectin domain family 11, member A) was very highly enriched in hucMSC-EVs. In addition, hucMSC-EVs enhanced the shift from adipogenic to osteogenic differentiation of BMSCs via delivering CLEC11A in vitro. Moreover, CLEC11A was required for the inhibitory effects of hucMSC-EVs on osteoclast formation. Conclusion: Our results suggest that hucMSC-EVs serve as a critical regulator of bone metabolism by transferring CLEC11A and may represent a potential agent for prevention and treatment of osteoporosis.

In elderly people particularly in postmenopausal women, inadequate bone formation by osteoblasts originating from bone marrow mesenchymal stem cells (BMSCs) for compensation of bone resorption by osteoclasts is a major reason for osteoporosis. Enhancing osteoblastic differentiation of BMSCs is a feasible therapeutic strategy for osteoporosis. Here, bone marrow stromal cell (ST)-derived exosomes (STExos) are found to remarkably enhance osteoblastic differentiation of BMSCs in vitro. However, intravenous injection of STExos is inefficient in ameliorating osteoporotic phenotypes in an ovariectomy (OVX)-induced postmenopausal osteoporosis mouse model, which may be because STExos are predominantly accumulated in the liver and lungs, but not in bone. Hereby, the STExo surface is conjugated with a BMSC-specific aptamer, which delivers STExos into BMSCs within bone marrow. Intravenous injection of the STExo-Aptamer complex enhances bone mass in OVX mice and accelerates bone healing in a femur fracture mouse model. These results demonstrate the efficiency of BMSC-specific aptamer-functionalized STExos in targeting bone to promote bone regeneration, providing a novel promising approach for the treatment of osteoporosis and fracture.

Osteoporosis is a debilitating bone disease affecting millions of people. Here, we used human urine-derived stem cells (USCs), which were noninvasively harvested from unlimited and easily available urine, as a “factory” to obtain extracellular vesicles (USC-EVs) and demonstrated that the systemic injection of USC-EVs effectively alleviates bone loss and maintains bone strength in osteoporotic mice by enhancing osteoblastic bone formation and suppressing osteoclastic bone resorption. More importantly, the anti-osteoporotic properties of USC-EVs are not notably disrupted by the age, gender, or health condition (with or without osteoporosis) of the USC donor. Mechanistic studies determined that collagen triple-helix repeat containing 1 (CTHRC1) and osteoprotegerin (OPG) proteins are enriched in USC-EVs and required for USC-EV-induced pro-osteogenic and anti-osteoclastic effects. Our results suggest that autologous USC-EVs represent a promising novel therapeutic agent for osteoporosis by promoting osteogenesis and inhibiting osteoclastogenesis by transferring CTHRC1 and OPG.

Objective: Senile osteoporosis is one of the most common age-related diseases worldwide. Accumulating evidences have indicated that young blood can reverse age-related impairments. Extracellular vesicles (EVs) exert therapeutic effects in a variety of diseases by delivering bioactive molecules such as microRNAs (miRNAs). The aim of the study is to evaluate the therapeutic potential of EVs from human umbilical cord blood plasma (UCB-EVs) on senile osteoporosis and to preliminarily clarify the underlying mechanism. Methods: UCB-EVs were injected into the tail vein of aged (16 months old) male C57BL/6 mice. Microcomputed tomography was performed to evaluate bone mass and microarchitecture of mice. The osteogenic and osteoclastic activities were determined by quantitative real-time PCR (qRT-PCR), histological examination and western blot analysis. in vitro, qRT-PCR assay was undertaken to explore the enrichment levels of a number of miRNAs that have positive effects in reducing bone loss. The efficacy of UCB-EVs on osteoblastic differentiation of bone marrow mesenchymal stromal cells (BMSCs) and osteoclastogenesis of RAW264.7 cells were assessed by cytochemical staining. Gene and protein expression changes were detected by qRT-PCR and western blotting respectively. Meanwhile, the roles of the selected miRNA in the regulatory effects of UCB-EVs on BMSCs and RAW264.7 cells were evaluated by using specific miRNA inhibitor. Results: The intravenous injection of UCB-EVs for two months attenuated bone loss in old mice, as defined by increased trabecular and cortical bone mass, enhanced osteoblast formation and reduced osteodast formation compared to the control mice. In vitro, UCB-EVs could promote the osteogenic differentiation of BMSCs and inhibit the osteoclastogenesis of RAW264.7 cells. Moreover, it was confirmed that miR-3960 was highly enriched in UCB-EVs and miR-3960 inhibitor reversed the stimulatory effect of UCB-LVs on osteoblastic differentiation of BMSCs. Conclusion: Our findings indicate that UCB-EVs ameliorate age-related bone loss by stimulating bone formation and inhibiting bone resorption, and miR-3960 mediated the osteogenic effect of UCB-EVs on BMSCs. Thus, UCB-EVs may represent a promising agent for prevention of senile osteoporosis. (C) 2019 Elsevier Inc. All rights reserved.

Background/Aims: Recently, we and others showed that the relative abundance of a specific vessel subtype, strongly positive for CD31 and Endomucin (CD31hiEmcnhi), is associated with bone formation and bone loss, and platelet-derived growth factor-BB (PDGF-BB) secreted by preosteoclasts induces the formation of the specific vessels and thereby stimulates osteogenesis. Inhibition of Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) has been shown to block the fusion of preosteoclasts into mature osteoclasts. However, it is unclear whether inhibition of SHP-2 could promote preosteoclast-induced angiogenesis and then enhance bone formation. This study aimed to determine the effects of a specific SHP-2 inhibitor (NSC-87877) on CD31 hiEmcnhi vessel and bone formation. Methods: 3-month-old C57BL/6 mice were subjected to either ovariectomy (OVX) or sham operation. OVX mice were intraperitoneally injected with NSC-87877 and the control (sham) mice were treated with an equal volume of diluents (PBS). Two months later, bone samples from mice were collected for µCT, histological, immunohistochemical and immunofluorescent analyses to assess bone mass, osteogenic and osteoclastic acitivities, as well as the densities of CD31hiEmcnhi vessels. A series of angiogenesis- related assays were performed to test the effects of NSC-87877 on the pro-angiogenic activities of preosteoclasts in vitro. Results: We found that NSC-87877 is sufficient to induce bone-sparing effects in OVX-induced osteoporotic mouse model. We also found that NSC-87877 induces higher numbers of preosteoclasts and CD31hiEmcnhi vessels and higher levels of PDGF-BB in bone marrow of osteoporotic mice. In vitro assays showed that NSC-87877 prevents preosteoclast fusion, increases PDGF-BB production, and augments the pro-angiogenic abilities of preosteoclasts. Conclusion: Our results suggest that NSC-87877 can be used as a promising therapeutic agent for osteoporosis by inhibiting osteoclast formation and promoting preosteoclast-induced angiogenesis.

Chronic non-healing wounds represent one of the most common complications of diabetes and need advanced treatment strategies. Exosomes are key mediators of cell paracrine action and can be directly utilized as therapeutic agents for tissue repair and regeneration. Here, we explored the effects of exosomes from human urine-derived stem cells (USC-Exos) on diabetic wound healing and the underlying mechanism. Methods: USCs were characterized by flow cytometry and multipotent differentiation potential analyses. USC-Exos were isolated from the conditioned media of USCs and identified by transmission electron microscopy and flow cytometry. A series of functional assays in vitro were performed to assess the effects of USC-Exos on the activities of wound healing-related cells. Protein profiles in USC-Exos and USCs were examined to screen the candidate molecules that mediate USC-Exos function. The effects of USC-Exos on wound healing in streptozotocin-induced diabetic mice were tested by measuring wound closure rates, histological and immunofluorescence analyses. Meanwhile, the role of the candidate protein in USC-Exos-induced regulation of angiogenic activities of endothelial cells and diabetic wound healing was assessed. Results: USCs were positive for CD29, CD44, CD73 and CD90, but negative for CD34 and CD45. USCs were able to differentiate into osteoblasts, adipocytes and chondrocytes. USC-Exos exhibited a cup- or sphere-shaped morphology with a mean diameter of 51.57 ± 2.93 nm and positive for CD63 and TSG101. USC-Exos could augment the functional properties of wound healing-related cells including the angiogenic activities of endothelial cells. USC-Exos were enriched in the proteins that are involved in regulation of wound healing-related biological processes. Particularly, a pro-angiogenic protein called deleted in malignant brain tumors 1 (DMBT1) was highly expressed in USC-Exos. Further functional assays showed that DMBT1 protein was required for USC-Exos-induced promotion of angiogenic responses of cultured endothelial cells, as well as angiogenesis and wound healing in diabetic mice. Conclusion: Our findings suggest that USC-Exos may represent a promising strategy for diabetic soft tissue wound healing by promoting angiogenesis via transferring DMBT1 protein.

Osteoporosis is a frequent complication of chronic inflammatory diseases and increases in the pro-inflammatory cytokines make an important contribution to bone loss by promoting bone resorption and impairing bone formation. Omentin-1 is a newly identified adipocytokine that has anti-inflammatory effects, but little is known about the role of omentin-1 in inflammatory osteoporosis. Here we generated global omentin-1 knockout ( omentin-1 −/− ) mice and demonstrated that depletion of omentin-1 induces inflammatory bone loss-like phenotypes in mice, as defined by abnormally elevated pro-inflammatory cytokines, increased osteoclast formation and bone tissue destruction, as well as impaired osteogenic activities. Using an inflammatory cell model induced by tumor necrosis factor-α (TNF-α), we determined that recombinant omentin-1 reduces the production of pro-inflammatory factors in the TNF-α-activated macrophages, and suppresses their anti-osteoblastic and pro-osteoclastic abilities. In the magnesium silicate-induced inflammatory osteoporosis mouse model, the systemic administration of adenoviral-delivered omentin-1 significantly protects from osteoporotic bone loss and inflammation. Our study suggests that omentin-1 can be used as a promising therapeutic agent for the prevention or treatment of inflammatory bone diseases by downregulating the pro-inflammatory cytokines.

The application of blood plasma for soft tissue wound healing is receiving much more attention recently. Exosomes are critical paracrine mediators that can be obtained from biological fluids including plasma and be able to induce regenerative effects by transferring bioactive molecules such as microRNAs (miRNAs). This study aimed to investigate the effects of exosomes from human umbilical cord blood plasma (UCB-Exos) on wound healing and to elucidate the underlying mechanism. Methods: UCB-Exos were isolated by ultracentrifugation and subcutaneously injected into full-thickness skin wounds in mice. The efficacy of UCB-Exos on wound healing was evaluated by measuring wound closure rates, histological analysis and immunofluorescence examinations. In vitro, quantitative real-time PCR (qRT-PCR) analysis was performed to detect the expression levels of a class of miRNAs that have positive roles in regulating wound healing. The scratch wound assay, transwell assay and cell counting kit-8 analysis were conducted to assess the effects of UCB-Exos on migration and proliferation of human skin fibroblasts and endothelial cells. Tube formation assay was carried out to test the impact of UCB-Exos on angiogenic tube formation ability of endothelial cells. Meanwhile, by using specific RNA inhibitors or siRNAs, the roles of the candidate miRNA and its target genes in UCB-Exos-induced regulation of function of fibroblasts and endothelial cells were assessed. Results: The local transplantation of UCB-Exos into mouse skin wounds resulted in accelerated re-epithelialization, reduced scar widths, and enhanced angiogenesis. In vitro, UCB-Exos could promote the proliferation and migration of fibroblasts, and enhance the angiogenic activities of endothelial cells. Notably, miR-21-3p was found to be highly enriched in UCB-Exos and served as a critical mediator in UCB-Exos -induced regulatory effects through inhibition of phosphatase and tensin homolog (PTEN) and sprouty homolog 1 (SPRY1). Conclusion: Our results suggest that UCB-Exos are important effectors of plasma activity and can be used as a novel promising strategy for soft tissue wound healing.

Recently, researchers identified a distinct vessel subtype called type H vessels that couple angiogenesis and osteogenesis. We previously found that type H vessels are reduced in ovariectomy (OVX)-induced osteoporotic mice, and preosteoclasts are able to secrete platelet-derived growth factor-BB (PDGF-BB) to stimulate type H vessel formation and thereby to promote osteogenesis. This study aimed to explore whether harmine, a β-carboline alkaloid, is capable of preventing bone loss in OVX mice by promoting preosteoclast PDGF-BB-induced type H vessel formation. METHODS:The impact of harmine on osteoclastogenesis of RANKL-stimulated RAW264.7 cells was verified by gene expression analysis and tartrate-resistant acid phosphatase (TRAP) staining. Enzyme-linked immunosorbent assay (ELISA) was conducted to test PDGF-BB production by preosteoclasts. A series of angiogenesis-related assays in vitro were performed to assess the pro-angiogenic effects of the conditioned media from RANKL-stimulated RAW264.7 cells treated with or without harmine. Meanwhile, the role of PDGF-BB in this process was determined. In vivo, OVX mice were intragastrically administrated with harmine emulsion or an equal volume of vehicle. 2 months later, bone samples were collected for µCT, histological, immunohistochemical and immunofluorescent analyses to evaluate bone mass, osteogenic and osteoclastic activities, as well as the numbers of type H vessels. Bone marrow PDGF-BB concentrations were assessed by ELISA. RESULTS:Exposure of RANKL-stimulated RAW264.7 cells to harmine enhanced the formation of preosteoclasts and the production of PDGF-BB. Harmine augmented the ability of RANKL-stimulated RAW264.7 cells to promote angiogenesis of endothelial cells, whereas the effect was blocked by PDGF-BB inhibition. In vivo, the oral administration of harmine emulsion to OVX mice resulted in enhanced trabecular bone mass and osteogenic responses, increased numbers of preosteoclasts, as well as reduced numbers of osteoclasts and fat cells. Moreover, OVX mice treated with harmine exhibited higher levels of bone marrow PDGF-BB and much more type H vessels in bone. CONCLUSION:Harmine may exert bone-sparing effects by suppression of osteoclast formation and promotion of preosteoclast PDGF-BB-induced angiogenesis.

Harmine, a β-carboline alkaloid, is widely distributed in the plants, animals as well as in human tissues and body fluids. Harmine has various types of pharmacological activities including antimicrobial, antiplasmodial, antioxidative, antitumor, antimutagenic, antidiabetic, vasorelaxant and central excitation properties. Moreover, it can exert regenerative and protective effects on bone and cartilage tissues by regulating the proliferation, differentiation and metabolism of osteoclasts, osteoblasts and chondrocytes. These features make harmine a novel candidate for the prevention and treatment of bone and cartilage diseases, such as osteoporosis, bone fracture and osteoarthritis.