High-mobility group box 1 accelerates distraction osteogenesis healing via the recruitment of endogenous stem/progenitor cells

Li X., Wang X., Zhang C., Wang J., Wang S., Hu L.

Evidences have suggested that the metabolic function is the key regulator to the fate of MSCs, but its function in senescence of MSC and the underlying mechanism is unclear. Therefore, the purpose of this study was to investigate the metabolic activity of MSCs and its possible mechanism during aging.We used the Seahorse XF24 Analyzer to understand OCR and ECAR in BMSCs and used RT-PCR to analyze the gene expression of mitochondrial biogenesis and key enzymes in glycolysis. We analyzed BMSC mitochondrial activity by MitoTracker Deep Red and JC-1 staining, and detected NAD+/NADH ratio and ATP levels in BMSCs. Microarray and proteomic analyses were performed to detect differentially expressed genes and proteins in BMSCs. The impact of aging on BMSCs through mitochondrial electron transport chain (ETC) was evaluated by Rotenone and Coenzyme Q10.Our results demonstrated that the oxidative phosphorylation and glycolytic activity of BMSCs in aged mice were significantly decreased when compared with young mice. BMSCs in aged mice had lower mitochondrial membrane potential, NAD+/NADH ratio, and ATP production than young mice. FABP4 may play a key role in BMSC senescence caused by fatty acid metabolism disorders.Taken together, our results indicated the dysfunction of the metabolic activity of BMSCs in aged mice, which would play the important role in the impaired biological properties. Therefore, the regulation of metabolic activity may be a potential therapeutic target for enhancing the regenerative functions of BMSCs.

Deng Z., Ren Y., Park M.S., Kim H.K.

In Legg-Calvé-Perthes disease (LCPD), a loss of blood supply to the juvenile femoral head leads to extensive cell death and release of damage-associated molecular patterns (DAMPs). Over time chronic inflammatory repair process is observed with impaired bone regeneration. Increased fibrous tissue and adipose tissue are seen in the marrow space with decreased osteogenesis in a piglet model of LCPD, suggesting inhibition of osteoblastic differentiation and stimulation of fibroblastic and adipogenic differentiation of mesenchymal stem cell (MSC) during the healing process. Little is known about the DAMPs present in the necrotic femoral head and their effects on MSC differentiation. The purpose of this study was to characterize the DAMPs present in the femoral head following ischemic osteonecrosis and to determine their effects on MSC differentiation. Necrotic femoral heads were flushed with saline at 48 h, 2 weeks and 4 weeks following the induction of ischemic osteonecrosis in piglets to obtain necrotic bone fluid (NBF). Western blot analysis of the NBF revealed the presence of prototypic DAMP, high mobility group box 1 (HMGB1), and other previously described DAMPs: biglycan, 4-hydroxynonenal (4-HNE), and receptor activator of NF-κB ligand (RANKL). ELISA of the NBF revealed increasing levels of inflammatory cytokines IL1β, IL6 and TNFα with the temporal progression of osteonecrosis. To determine the effects of NBF on MSC differentiation, we cultured primary porcine MSCs with NBF obtained by in vivo necrotic bone flushing method. NBF inhibited osteoblastic differentiation of MSCs with significantly decreased OSX expression ( p = 0.008) and Von Kossa/Alizarin Red staining for mineralization. NBF also significantly increased the expression of proliferation markers Ki67 ( p = 0.03) and PCNA ( p < 0.0001), and fibrogenic markers Vimentin ( p = 0.02) and Fibronectin ( p = 0.04). Additionally, NBF treated MSC cells showed significantly elevated RANKL/OPG secretion ratio ( p = 0.003) and increased expression of inflammatory cytokines IL1β ( p = 0.006) and IL6 ( p < 0.0001). To specifically assess the role of DAMPs in promoting the fibrogenesis, we treated porcine fibroblasts with artificial NBF produced by bone freeze-thaw method. We found increased fibroblastic cell proliferation in an NBF dose-dependent manner. Lastly, we studied the effect of HMGB1, a prototypic DAMP, and found that HMGB1 partially contributes to MSC proliferation and fibrogenesis. In summary, our findings show that DAMPs and the inflammatory cytokines present in the necrotic femoral head inhibit osteogenesis and promote fibrogenesis of MSCs, potentially contributing to impaired bone regeneration following ischemic osteonecrosis as observed in LCPD. • NBF from necrotic femoral head contained DAMPs and inflammatory cytokines. • The content of NBF changed over time mediating a chronic inflammatory response. • NBF decreased osteogenesis and increased fibrogenesis of MSCs.

Wang X., Jiang H., Guo L., Wang S., Cheng W., Wan L., Zhang Z., Xing L., Zhou Q., Yang X., Han H., Chen X., Wu X.

Cell-based therapeutics bring great hope in areas of unmet medical needs. Mesenchymal stem cells (MSCs) have been suggested to facilitate neovascularization mainly by paracrine action. Endothelial progenitor cells (EPCs) can migrate to ischemic sites and participate in angiogenesis. The combination cell therapy that includes MSCs and EPCs has a favorable effect on ischemic limbs. However, the mechanism of combination cell therapy remains unclear. Herein, we investigate whether stromal cell-derived factor (SDF)-1 secreted by MSCs contributes to EPC migration to ischemic sites via CXCR4/Phosphoinositide 3-Kinases (PI3K)/protein kinase B (termed as AKT) signaling pathway. First, by a “dual-administration” approach, intramuscular MSC injections were supplemented with intravenous Qdot® 525 labeled-EPC injections in the mouse model of hind limb ischemia. Then, the mechanism of MSC effect on EPC migration was detected by the transwell system, tube-like structure formation assays, western blot assays in vitro. Results showed that the combination delivery of MSCs and EPCs enhanced the incorporation of EPCs into the vasculature and increased the capillary density in mouse ischemic hind limb. The numbers of CXCR4-positive EPCs increased after incubation with MSC-conditioned medium (CM). MSCs contributed to EPC migration and tube-like structure formation, both of which were suppressed by AMD3100 and wortmannin. Phospho-AKT induced by MSC-CM was attenuated when EPCs were pretreated with AMD3100 and wortmannin. In conclusion, we confirmed that MSCs contributes to EPC migration, which is mediated via CXCR4/PI3K/AKT signaling pathway.

Bouland C., Philippart P., Dequanter D., Corrillon F., Loeb I., Bron D., Lagneaux L., Meuleman N.

Bone regeneration is a complex, well-orchestrated process based on the interactions between osteogenesis and angiogenesis, observed in both physiological and pathological situations. However, specific conditions (e.g., bone regeneration in large quantity, immunocompromised regenerative process) require additional support. Tissue engineering offers novel strategies. Bone regeneration requires a cell source, a matrix, growth factors and mechanical stimulation. Regenerative cells, endowed with proliferation and differentiation capacities, aim to recover, maintain, and improve bone functions. Vascularization is mandatory for bone formation, skeletal development, and different osseointegration processes. The latter delivers nutrients, growth factors, oxygen, minerals, etc. The development of mesenchymal stromal cells (MSCs) and endothelial progenitor cells (EPCs) cocultures has shown synergy between the two cell populations. The phenomena of osteogenesis and angiogenesis are intimately intertwined. Thus, cells of the endothelial line indirectly foster osteogenesis, and conversely, MSCs promote angiogenesis through different interaction mechanisms. In addition, various studies have highlighted the importance of the microenvironment via the release of extracellular vesicles (EVs). These EVs stimulate bone regeneration and angiogenesis. In this review, we describe (1) the phenomenon of bone regeneration by different sources of MSCs. We assess (2) the input of EPCs in coculture in bone regeneration and describe their contribution to the osteogenic potential of MSCs. We discuss (3) the interaction mechanisms between MSCs and EPCs in the context of osteogenesis: direct or indirect contact, production of growth factors, and the importance of the microenvironment via the release of EVs.

Knecht R.S., Bucher C.H., Van Linthout S., Tschöpe C., Schmidt-Bleek K., Duda G.N.

A misdirected or imbalanced local immune composition is often one of the reasons for unsuccessful regeneration resulting in scarring or fibrosis. Successful healing requires a balanced initiation and a timely down-regulation of the inflammation for the re-establishment of a biologically and mechanically homeostasis. While biomaterial-based approaches to control local immune responses are emerging as potential new treatment options, the extent to which biophysical material properties themselves play a role in modulating a local immune niche response has so far been considered only occasionally. The communication loop between extracellular matrix, non-hematopoietic cells, and immune cells seems to be specifically sensitive to mechanical cues and appears to play a role in the initiation and promotion of a local inflammatory setting. In this review, we focus on the crosstalk between ECM and its mechanical triggers and how they impact immune cells and non-hematopoietic cells and their crosstalk during tissue regeneration. We realized that especially mechanosensitive receptors such as TRPV4 and PIEZO1 and the mechanosensitive transcription factor YAP/TAZ are essential to regeneration in various organ settings. This indicates novel opportunities for therapeutic approaches to improve tissue regeneration, based on the immune-mechanical principles found in bone but also lung, heart, and skin.

Feng Y., Hu S., Liu L., Ke J., Long X.

Background High mobility group 1 protein (HMGB1) is related with inflammation. Our former research reported that substantial HMGB1 situates at the synovium of osteoarthritis of temporomandibular joint (TMJOA) patients. Objective This study investigated whether HMGB1 promotes synovial angiogenesis of TMJOA and its underlying mechanism. Methods Human synovial fibroblasts were stimulated with HMGB1, the expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible transcription factor-1α (HIF-1α) in these cells was explored by Western blotting, real-time PCR and immunofluorescent staining. The angiogenic capacity of these cells was assayed by tube formation and cell migration of human umbilical vein endothelial cells (HUVECs). The specific inhibitor against HMGB1, VEGF, Erk or JNK was added in these cells, respectively. Complete Freund's adjuvant (CFA)-induced TMJOA rats was produced. The changes in their synovium and synovial fluid were detected by immunofluorescent staining and ELISA. Results HMGB1 effectively up-regulated the production of VEGF and HIF-1α in TMJOA synovial fibroblasts through the activation of Erk and JNK. Conditioned medium from HMGB1-treated TMJOA synovial fibroblasts significantly promoted tube formation and migration in HUVECs, while attenuated those after the addition of certain inhibitor for VEGF. Furthermore, the specific inhibitor against HMGB1 vanished the neovascularization and production of HIF-1α, VEGF and CD34 in the synovium of rat TMJOA induced by CFA injection. Additionally, this inhibitor led to the reduction of IL-6, IL-1β and TNF-α in the synovial fluid of those rats. Conclusion These findings disclose a key role for HMGB1 in governing synovial angiogenesis and as a therapeutic target against TMJOA.

Lan J., Luo H., Wu R., Wang J., Zhou B., Zhang Y., Jiang Y., Xu J.

Objective: In patients with peripheral artery disease, blockages in arterioles <1 mm cannot be treated surgically, and there are currently few effective medicines. Studies have shown that inflammation in ischemic tissue is related to injury recovery and angiogenesis, but insufficient attention has been paid to this area. Studies have suggested that HMGB1 (high mobility group protein 1), which is released by ischemic tissue, promotes angiogenesis, but the mechanism is not entirely clear. In this study, we tested the internalization of HMGB1 in endothelial cells and investigated a novel proangiogenic pathway. Approach and Results: Using green fluorescent protein–tagged HMGB1 to stimulate endothelial cells, we demonstrated HMGB1 internalization via dynamin and RAGE (receptor for advanced glycation end products)-dependent signaling. Using a fluorescence assay, we detected internalized protein fusion to lysosomes, followed by activation of CatB (cathepsin B) and CatL (cathepsin L). The latter promoted the release of VEGF (vascular endothelial growth factor)-A and endoglin and upregulated the capacities of cell migration, proliferation, and tube formation in endothelial cells. We identified that the cytokine-induced fragment—a key functional domain in HMGB1—mediates the internalization and angiogenic function of HMGB1. We further confirmed that HMGB1 internalization also occurs in vivo in endothelial cells and promotes angiogenesis in mouse femoral artery ligation. Conclusions: In this study, we identified a novel pathway of HMGB1 internalization–induced angiogenesis in endothelial cells. This finding sheds light on the regulatory role of inflammatory factors in angiogenesis through cell internalization and opens a new door to understand the relationship between inflammation and angiogenesis in ischemic diseases.

Bianchi M.E., Mezzapelle R.

The CXCR4 receptor upon binding its ligands triggers multiple signaling pathways that orchestrate cell migration, hematopoiesis and cell homing and retention in the bone marrow. However, CXCR4 also directly controls cell proliferation of non-hematopoietic cells. This review focuses on recent reports pointing to its pivotal role in tissue regeneration and stem cell activation, and discusses the connection to the known role of CXCR4 in promoting tumor growth. The mechanisms may be similar in all cases, since regeneration often recapitulates developmental processes, and cancer often exploits developmental pathways. Moreover, cell migration and cell proliferation appear to be downstream of the same signaling pathways. A deeper understanding of the complex signaling originating from CXCR4 is needed to exploit the opportunities to repair damaged organs safely and effectively.

Goto T., Miyagawa S., Tamai K., Matsuura R., Kido T., Kuratani T., Shimamura K., Sakaniwa R., Harada A., Sawa Y.

Objectives High-mobility group box 1 protein (HMGB1) fragment enhances bone marrow-derived mesenchymal stem cell (BM-MSC) recruitment to damaged tissue to promote tissue regeneration. This study aimed to evaluate whether systemic injection of HMGB1 fragment could promote tissue repair in a rat model of myocardial infarction (MI). Methods HMGB1 (n = 14) or phosphate buffered saline (n = 12, control) was administered to MI rats for 4 days. Cardiac performance and left ventricular remodeling were evaluated using ultrasonography and immunostaining. BM-MSC recruitment to damaged tissue in green fluorescent protein-bone marrow transplantation (GFP-BMT) models was evaluated using immunostaining. Results At four weeks post-treatment, the left ventricular ejection fraction was significantly improved in the HMGB1 group compared to that in the control. Interstitial fibrosis and cardiomyocyte hypertrophy were also significantly attenuated in the HMGB1 group compared to the control. In the peri-infarction area, VEGF-A mRNA expression was significantly higher and TGFβ expression was significantly attenuated in the HMGB1 group than in the control. In GFP-BMT rats, GFP+/PDGFRα+ cells were significantly mobilized to the peri-infarction area in the HMGB1 group compared to that in the control, leading to the formation of new vasculature. In addition, intravital imaging revealed that more GFP+/PDGFRα+ cells were recruited to the peri-infarction area in the HMGB1 group than in the control 12 h after treatment. Conclusions Systemic administration of HMGB1 induced angiogenesis and reduced fibrosis by recruiting PDGFRα+ mesenchymal cells from the bone marrow, suggesting that HMGB1 administration might be a new therapeutic approach for heart failure after MI.

Zhang F., Huan L., Xu T., Li G., Zheng B., Zhao H., Guo Y., Shi J., Sun J., Chen A.

Mechanical stress has been recognized as a key inducer of bone regeneration in bone damage, which is experimentally mimicked by distraction osteogenesis (DO), a bone-regenerative process induced by post-osteotomy distraction of the surrounding vascularized bone segments, and realized by new bone formation within the distraction gap. The mechanisms that underlie the DO-induced bone regeneration remain poorly understood and a role of macrophages in the process has been inadequately studied. Here, in a mouse model of DO, we showed significant increase in macrophages in the regeneration area. Moreover, in a loss-of-function approach by depleting inflammatory macrophages, the bone regeneration was compromised by assessment of histology and molecular biology. Thus, our study demonstrates the necessary participation of inflammatory macrophages in the process of DO-induced bone regeneration, and suggests that targeting inflammatory macrophages may help to improve clinical bone repair.

Jin S., He D., Luo D., Wang Y., Yu M., Guan B., Fu Y., Li Z., Zhang T., Zhou Y., Wang C., Liu Y.

The host immune response to bone biomaterials is vital in determining scaffold fates and bone regeneration outcomes. The nanometer-scale interface of biomaterials, which independently controls physical inputs to cells, regulates osteogenic differentiation of stem cells and local immune response. Herein, we fabricated biomimetic hierarchical intrafibrillarly mineralized collagen (HIMC) with a bone-like staggered nanointerface and investigated its immunomodulatory properties and mesenchymal stem cell (MSC) recruitment during endogenous bone regeneration. The acquired HIMC potently induced neo-bone formation by promoting CD68+CD163+ M2 macrophage polarization and CD146+STRO-1+ host MSC recruitment in critical-sized bone defects. Mechanistically, HIMC facilitated M2 macrophage polarization and interleukin (IL)-4 secretion to promote MSC osteogenic differentiation. An anti-IL4 neutralizing antibody significantly reduced M2 macrophage-mediated osteogenic differentiation of MSCs. Moreover, HIMC-loaded-IL-4 implantation into critical-sized mandible defects dramatically enhanced bone regeneration and CD68+CD163+ M2 macrophage polarization. The depletion of monocyte/macrophages by clodronate liposomes significantly impaired bone regeneration by HIMC, but did not affect MSC recruitment. Thus, in emulating natural design, the hierarchical nanointerface possesses the capacity to recruit host MSCs and promote endogenous bone regeneration by immunomodulation of macrophage polarization through IL-4.

Josephson A.M., Bradaschia-Correa V., Lee S., Leclerc K., Patel K.S., Muinos Lopez E., Litwa H.P., Neibart S.S., Kadiyala M., Wong M.Z., Mizrahi M.M., Yim N.L., Ramme A.J., Egol K.A., Leucht P.

Significance As we age, our capacity for tissue repair and regeneration in response to injury declines. Accordingly, bone repair is delayed and impaired in older patients. At the cornerstone of bone healing is the skeletal stem/progenitor cell (SSPC), whose function and number diminishes with age. However, the mechanisms driving this decline remain unclear. Here, we identify age-associated inflammation (“inflamm-aging”) as the main culprit of SSPC dysfunction and provide support for a central role of NF-κB as a mediator of inflamm-aging. Our results show that modification of the inflammatory environment may be a translational approach to functionally rejuvenate the aged SSPC, thereby improving the regenerative capacity of the aged skeleton.

Da'at Arina Y., Rubianto M., Ferdiansyah F., Sudiana I., Rahayu R., Notobroto H.

Andersson U., Yang H., Harris H.

Alarmins are preformed, endogenous molecules that can be promptly released to signal cell or tissue stress or damage. The ubiquitous nuclear molecule high-mobility group box 1 protein (HMGB1) is a prototypical alarmin activating innate immunity. HMGB1 serves a dual alarmin function. The protein can be emitted to alert adjacent cells about endangered homeostasis of the HMGB1-releasing cell. In addition to this expected path of an alarmin, extracellular HMGB1 can be internalized via RAGE-receptor mediated endocytosis to the endolysosomal compartment while attached to other extracellular proinflammatory molecules. The endocytosed HMGB1 may subsequently destabilize lysosomal membranes. The HMGB1-bound partner molecules depend on the HMGB1-mediated transport and the induced lysosomal leakage to obtain access to endosomal and cytosolic reciprocal sensors to communicate extracellular threat and to initiate the proper activation pathways.

Xue X., Chen X., Fan W., Wang G., Zhang L., Chen Z., Liu P., Liu M., Zhao J.

High-mobility group box 1 (HMGB1) facilitates neural stem cells (NSCs) proliferation and differentiation into neuronal linage. However, the effect of HMGB1 on NSCs migration is still elusive. The present study is to investigate the corelation between HMGB1 and NSCs migration and the potential mechanism. The results indicated that 1 ng/ml HMGB1 promoted NSCs proliferation using CCK8 assays. Moreover, data showed that 1 ng/ml HMGB1 facilitated NSCs migration via filopodia formation using phase-contrast and transwell assays. Furthermore, 1 ng/ml HMGB1 upregulated the expression of RAGE, one of the HMGB1 receptor, using western blotting assays and immunofluorescence staining. In addition, 1 ng/ml HMGB1 increased the percentage of filopodia formation using phalloidin staining. Meanwhile, the enhanced migration effect could be abrogated by 50 nM FPS-ZM1, one of the RAGE antagonist, and RAGE-specific siRNA through immunofluorescence and phalloidin staining. Together, our data demonstrate that HMGB1/RAGE axis facilitates NSCs migration via promoting filopodia formation, which might serve as a candidate for central nervous system (CNS) injury treatment and/or a preconditioning method for NSCs implantation.

Ruggieri E., Domenico E.D., Locatelli A.G., Isopo F., Damanti S., Lorenzo R.D., Milan E., Musco G., Rovere-Querini P., Cenci S., Vénéreau E.

Ren Y., Zhu D., Han X., Zhang Q., Chen B., Zhou P., Wei Z., Zhang Z., Cao Y., Zou H.

HMGB1 that belongs to the High Mobility Group-box superfamily, is a nonhistone chromatin associated transcription factor. It is present in the nucleus of eukaryotes and can be actively secreted or passively released by kinds of cells. HMGB1 is important for maintaining DNA structure by binding to DNA and histones, protecting it from damage. It also regulates the interaction between histones and DNA, affecting chromatin packaging, and can influence gene expression by promoting nucleosome sliding. And as a DAMP, HMGB1 binding to RAGE and TLRs activates NF-κB, which triggers the expression of downstream genes like IL-18, IL-1β, and TNF-α. HMGB1 is known to be involved in numerous physiological and pathological processes. Recent studies have demonstrated the significance of HMGB1 as DAMPs in the female reproductive system. These findings have shed light on the potential role of HMGB1 in the pathogenesis of diseases in female reproductive system and the possibilities of HMGB1-targeted therapies for treating them. Such therapies can help reduce inflammation and metabolic dysfunction and alleviate the symptoms of reproductive system diseases. Overall, the identification of HMGB1 as a key player in disease of the female reproductive system represents a significant breakthrough in our understanding of these conditions and presents exciting opportunities for the development of novel therapies.