Extravasated erythrocytes in cerebrospinal fluid (CSF) critically contribute to the pathogenesis of subarachnoid hemorrhage (SAH). Meningeal lymphatics have been reported to drain macromolecules and immune cells from CSF into cervical lymph nodes (CLNs). However, whether meningeal lymphatics are involved in clearing extravasated erythrocytes in CSF after SAH remains unclear. Here we show that a markedly higher number of erythrocytes are accumulated in the lymphatics of CLNs and meningeal lymphatics after SAH. When the meningeal lymphatics are depleted in a mouse model of SAH, the degree of erythrocyte aggregation in CLNs is significantly lower, while the associated neuroinflammation and the neurologic deficits are dramatically exacerbated. In addition, during SAH lymph flow is increased but without significant lymphangiogenesis and lymphangiectasia. Taken together, this work demonstrates that the meningeal lymphatics drain extravasated erythrocytes from CSF into CLNs after SAH, while suggesting that modulating this draining may offer therapeutic approaches to alleviate SAH severity.

Objective To investigate if enhancement of joint lymphangiogenesis by injecting VEGF-C adeno-associated virus (AAV) into joints has therapeutic efficacy in chronic inflammatory arthritis in mice. Methods TNF transgenic (TNF-Tg) mice were used as a model of chronic inflammatory arthritis. Human VEGF-C was cloned into an AAV expression vector to generate AAV-VEGF-C. AAV-VEGF-C or control AAV-Luc was injected into joints of TNF-Tg mice. MRI and lymphatic imaging were used during the 4-months following injection to assess changes in synovial volume and lymph flow from joint tissues to local draining lymph nodes. Joint inflammation, bone erosion and cartilage loss were examined by histologic analyses. Lymphatic vessel formation was assessed using immunohistochemistry. Results Intra-articular administration of AAV-VEGF-C virus significantly attenuated the increase in synovial volume and increased lymphatic vessel number in joint sections compared to AAV-Luc virus during the 4-month-period. This accompanied by reduced inflammation area, bone erosion, cartilage loss, and osteoclast numbers. Lymph flow from joints to local draining lymph nodes was slower in TNF-Tg mice than in wild-type littermates and was significantly improved with AAV-VEGF-C treatment. Conclusion Intra-articular injection of AAV-VEGF-C increased lymphangiogenesis and improved lymphatic drainage from inflamed joints, resulting in attenuation of joint tissue damage. Thus, improvement of joint lymphatic function by local administration of lymphatic growth factors represents a new therapeutic approach for chronic inflammatory arthritis.

Critical-sized bone defect repair in patients with diabetes mellitus remains a challenge in clinical treatment because of dysfunction of macrophage polarization and the inflammatory microenvironment in the bone defect region. Three-dimensional (3D) bioprinted scaffolds loaded with live cells and bioactive factors can improve cell viability and the inflammatory microenvironment and further accelerating bone repair. Here, we used modified bioinks comprising gelatin, gelatin methacryloyl (GelMA), and 4-arm poly (ethylene glycol) acrylate (PEG) to fabricate 3D bioprinted scaffolds containing BMSCs, RAW264.7 macrophages, and BMP-4-loaded mesoporous silica nanoparticles (MSNs). Addition of MSNs effectively improved the mechanical strength of GelMA/gelatin/PEG scaffolds. Moreover, MSNs sustainably released BMP-4 for long-term effectiveness. In 3D bioprinted scaffolds, BMP-4 promoted the polarization of RAW264.7 to M2 macrophages, which secrete anti-inflammatory factors and thereby reduce the levels of pro-inflammatory factors. BMP-4 released from MSNs and BMP-2 secreted from M2 macrophages collectively stimulated the osteogenic differentiation of BMSCs in the 3D bioprinted scaffolds. Furthermore, in calvarial critical-size defect models of diabetic rats, 3D bioprinted scaffolds loaded with MSNs/BMP-4 induced M2 macrophage polarization and improved the inflammatory microenvironment. And 3D bioprinted scaffolds with MSNs/BMP-4, BMSCs, and RAW264.7 cells significantly accelerated bone repair. In conclusion, our results indicated that implanting 3D bioprinted scaffolds containing MSNs/BMP-4, BMSCs, and RAW264.7 cells in bone defects may be an effective method for improving diabetic bone repair, owing to the direct effects of BMP-4 on promoting osteogenesis of BMSCs and regulating M2 type macrophage polarization to improve the inflammatory microenvironment and secrete BMP-2.

BackgroundIn this study, we sought to determine the cellular source of inducible nitric oxide synthase (iNOS) induced in lymphatic endothelial cells (LECs) in response to tumor necrosis factor (TNF), the effects of iNOS on lymphatic smooth muscle cell (LSMC) function and on the development of arthritis in TNF-transgenic (TNF-Tg) mice, and whether iNOS inhibitors improve lymphatic function and reduce joint destruction in inflammatory erosive arthritis.MethodsWe used quantitative polymerase chain reactions, immunohistochemistry, histology, and near-infrared imaging to examine (1) iNOS expression in podoplanin + LECs and lymphatic vessels from wild-type (WT) and TNF-Tg mice, (2) iNOS induction by TNF in WT LECs, (3) the effects of iNOS inhibitors on expression of functional muscle genes in LSMCs, and (4) the effects of iNOS inhibitors on lymphatic vessel contraction and drainage, as well as the severity of arthritis, in TNF-Tg mice.ResultsLECs from TNF-Tg mice had eight fold higher iNOS messenger RNA levels than WT cells, and iNOS expression was confirmed immunohistochemically in podoplanin + LECs in lymphatic vessels from inflamed joints. TNF (0.1 ng/ml) increased iNOS levels 40-fold in LECs. LSMCs cocultured with LECs pretreated with TNF had reduced expression of functional muscle genes. This reduction was prevented by ferulic acid, which blocked nitric oxide production. Local injection of L-N6-(1-iminoethyl)lysine 5-tetrazole-amide into inflamed paws of TNF-Tg mice resulted in recovery of lymphatic vessel contractions and drainage. Treatment of TNF-Tg mice with ferulic acid reduced synovial inflammation as well as cartilage and bone erosion, and it also restored lymphatic contraction and drainage.ConclusionsiNOS is produced primarily by LECs in lymphatic vessel efferent from inflamed joints of TNF-Tg mice in response to TNF and inhibits LSMC contraction and lymph drainage. Ferulic acid represents a potential new therapy to restore lymphatic function and thus improve inflammatory arthritis by inhibiting local production of nitric oxide by LSMCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-0963-8) contains supplementary material, which is available to authorized users.

In rat endplate chondrocytes the PI3K pathway mainly transduces IGF-1 effect on col2 expression while the ERK pathway mediates IGF-1 effect on MMP-13 expression.

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disorder, characterized by an increased number of M1-like macrophages in the joints. Polyphyllin I (PPI), one of the main components in the Rhizoma of Paris polyphyllin, displays a selective inhibitory effect on various tumor cells. Here we sought to investigate the anti-rheumatoid arthritis effects and mechanisms of PPI on macrophages in vivo and in vitro.Materials and Methods: In vitro, primary bone marrow-derived macrophages (BMMs) and peritoneal elucidated macrophages (PEMs) were stimulated by lipopolysaccharide (LPS) and Interferon (IFN)-γ and then treated with PPI. We determined the degree of activation of IKKα/β and p65, two key mediators of the NF-κB-mediated inflammatory pathway, by measuring their phosphorylated forms by Western blot. The p65 nuclear localization was detected by immunofluorescent staining. Further, a NF-κB-linked luciferase reporter plasmid, as well as those expressing key mediators of the Toll-like receptor 4 pathway, such as myeloid differentiation primary response 88 (MYD88), interleukin-1 receptor (IL-1R) associated kinase (IRAK)-1, TNF receptor associated factors (TRAF)-6, Transforming growth factor-b–activated kinase 1 (TAK1) and p65, were used to identify the mechanism by which PPI achieves its inhibitory effects on macrophage-mediated inflammation. Moreover, a NF-κB inhibitor, p65-targeted siRNAs, and a p65 plasmid were further used to validate the anti-inflammatory mechanism of PPI. In vivo, PPI (1 mg/kg) was administered intragastrically one time a day for 7 weeks starting on the 42nd day after the first immunization with collagen in a collagen-induced arthritis (CIA) mouse model. Micro-computed Tomography scanning, histological examination, F4/80 and iNOS double immunofluorescent staining and CD4 immunohistochemical staining were performed to determine the effect of PPI treatment on joint structure and inflammation in this model.Results: PPI reduced the inflammatory cytokines production of PEMs stimulated by LPS/IFN-γ, inhibited the phosphorylation of IKKα/β and p65, and prevented p65 nuclear localization. The NF-κB luciferase assay showed that the target of PPI was closely related to the NF-κB pathway. Moreover, NF-κB inhibition, siRNA-mediated knockdown of p65, and p65 overexpression eliminated PPI's inhibitory effect. In addition, PPI attenuated the bone erosion and synovitis, as well as M1-like macrophage and T cell infiltration, in the ankle joint of the CIA model.Conclusion: PPI demonstrated effective amelioration of synovial inflammation in the ankle joint of CIA mice while suppressing NF-κB-mediated production of pro-inflammatory effectors in activated macrophages.

Objective To investigate the distribution and alteration of lymphatic vessels and draining function in knee joints of normal and osteoarthritic mice. Methods For the mouse models of osteoarthritis (OA), we used mice with meniscal-ligamentous injury or mice with conditional knockout of the gene for cartilage transforming growth factor β (TGF β) type II receptor. The severity of cartilage loss and joint destruction was assessed histologically. Capillary and mature lymphatic vessels were identified and analyzed using double immunofluorescence staining and a whole-slide digital imaging system. Lymphatic drainage of knee joints was examined using near-infrared lymphatic imaging. Patient joint specimens obtained during total knee or hip arthroplasty were evaluated to verify the content validity of the mouse findings. Results Lymphatic vessels were distributed in soft tissues (mainly around the joint capsule, ligaments, fat pads, and muscles of normal knees). The number of lymphatic vessels, particularly the number of capillaries, was significantly increased in joints of mice with mild OA, while the number of mature lymphatic vessels was markedly decreased in joints of mice with severe OA. OA knees exhibited significantly decreased lymph clearance. The number of both capillary and mature lymphatic vessels was significantly decreased in the joints of patients with OA. Conclusion The whole-slide digital imaging system is a powerful tool, enabling the identification and assessment of lymphatic microvasculature in the entire mouse knee. Lymphatic capillaries and mature vessels are present in various soft tissues around articular spaces. Abnormalities of lymphatic vessels and draining function, including significantly reduced numbers of mature vessels and impaired clearance, are present in OA joints.

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