Hua Liao

Effects of Nitric Oxide on Notexin-Induced Muscle Inflammatory Responses

Excessive inflammatory response may delay the regeneration and damage the normal muscle fibers upon myoinjury. It would be important to be able to attenuate the inflammatory response and decrease inflammatory cells infiltration in order to improve muscle regeneration formation, resulting in better muscle functional recovery after myoinjury. This study was undertaken to explore the role of Nitric oxide (NO) during skeletal muscle inflammatory process, using a mouse model of Notexin induced myoinjury. Intramuscular injection (tibialis anterior, TA) of Notexin was performed for preparing mice myoinjury. NO synthase inhibitor (L-NAME) or NO donor (SNP) was intraperitoneally injected into model mice. On day 4 and 7 post-injury, expression of muscle-autoantigens and toll-like receptors (TLRs) was evaluated from muscle tissue by qRT-PCR and Western Blot; the intramuscular infiltration of monocytes/macrophage (CD11b+ or F4/80+ cells), CD8+ T cell (CD3ε+CD8α+), apoptotic cell (CD11b+caspase3+), and MHC-I molecule H-2Kb-expressing myofibers in damaged muscle were assessed by imunoflourecence analysis; the mRNAs expression of cytokines and chemokines associated with the preferential biological role during the muscle damage-induced inflammation response, were assessed by qRT-PCR. We detected the reduced monocytes/macrophages infiltration, and increased apoptotic cells in the damaged muscle treated with SNP comparing to untreatment. As well, SNP treatment down-regulated mRNA and protein levels of muscle autoantigens, TLR3, and mRNA levels of TNF-α, IL-6, MCP-1, MCP-3, and MIP-1α in damaged muscle. On the contrary, L-NAME induced more severe intramuscular infiltration of inflammatory cells, and mRNA level elevation of the above inflammatory mediators. Notably, we observed an increased number of MHC-I (H2-Kb) positive new myofibers, and of the infiltrated CD8+ T cells in damaged muscle at the day 7 after L-NAME treatment. The result herein shows that, NO can act as an endogenous anti-inflammatory molecule during the ongoing muscle inflammation. Our finding may provide new insight to optimize NO-based therapies for improving muscle regeneration after myoinjury.

PNIPAM hydrogel induces skeletal muscle inflammation response

Although researchers believe hydrogels have favorable biocompatibility in vitro and in vivo, these kinds of synthetic polymers still have different immunogenic behavior in vivo, and bring forth a long-lasting inflammatory response. In this study, we attempt to explore the possibility of intramuscular implantation of a specific PNIPAM hydrogel to induce a chronic inflammatory-myoinjury model in mice. We injected a temperature-sensitive PNIPAM hydrogel mixed with or without fast-type skeletal muscle C protein, into the tibialis anterior muscle (TA) of WT B6 mice, and analyzed mononuclear cell infiltration, muscle autoantigens and TLRs expression, and the intramuscular CD8+ T cells infiltration and MHC-I expression on the surface of the new myofiber in TA muscle on day 15, 30 and 60 post-injection. We found that the PNIPAM hydrogel induces a skeletal muscle inflammation over 2 months. The inflammation response in the hydrogel injection area reached the peak on day 30, and then gradually decreased. Interestingly, the mixture of PNIPAM hydrogel/C protein initiated a more severe and persistent muscle inflammatory response, than hydrogel, or C protein injection alone. Immunofluorescence analysis demonstrated that the hydrogel induced chronic muscle inflammation featured by the CD8+ T cell infiltration and increased class I MHC-positive regenerating myofibers. Our work holds new promise for preparing an animal model of muscle inflammation using hydrogel as a bio-driver. The hydrogel induced-myoinjury model may be useful for investigating pathologic mechanisms of muscle injury, inflammation and regeneration.

Inflammatory and immuno-reactivity in mice induced by intramuscular implants of HSNGLPL peptide grafted-polyurethane

Synthetic peptide-based polyurethanes (PUs), introduced as bioactive agents and possessing impressive properties, have emerged as attractive functional biomaterials for tissue regeneration. In this study, we developed a PU with a pendent HSNGLPL group through click reaction, which has strong affinity to TGF-β1. The peptide grafted-PUs, or PUs with BDO as the chain extender (control), were implanted into the gastrocnemius muscle (GN) of C57BL/6 mice, for evaluating their inflammatory and immuno-reactivity in vivo. We show herein that, after muscle implantation, BDO-PU induced a conspicuous monocyte/macrophage infiltration and myofiber degeneration. The inflammatory invasion and myofiber necrosis were mainly detected in the site around, but not far from, the implants, suggesting that the degraded PU matrix only triggers a local and limited inflammation in vivo. In contrast, peptide grafted-PU induced intramuscular inflammation was more complex and was sustained for more than 2 months. Apart from nonspecific monocyte/macrophage infiltration as in the case of BDO-PU, CD4+ T cells and dendritic cells (DCs), the members of the adaptive immune system, can be detected within the inflammatory site around peptide grafted-PU implants. The number of apoptotic macrophages in muscle containing peptide-PU was significantly lower compared to that in muscle containing BDO-PU. Thus, our present results suggest that, the PU matrix degradation-produced local environment is toxic to muscle cells and induces muscle degeneration. Moreover, highly aggregated peptide on PU might act as an immunogen to trigger intramuscular inflammation and lead to the delayed inflammatory response.

Polyurethane conjugating TGF-β on surface impacts local inflammation and endoplasmic reticulum stress in skeletal muscle.

The synthesized short peptide-polymers would provide key functions for tissue regeneration and repair, through enriching bioactive molecules on polymers or releasing these molecules pre-conjugated on the materials. We have developed a degradable polyurethane (PU) bearing HSNGLPL peptide, which has affinity binding ability to transforming growth factor-betas (TGF-β). For deeply understanding spatial release of TGF-β from the PU polymers and its localized bioactivity, quartz crystal microbalance (QCM) and Elisa test were used to verify TGF-β binding capacities in vitro and in vivo. The PU polymers, with or without pre-conjugating of TGF-β, were implanted into gastronomies muscle (GN) of C57BL/6 mice, for addressing TGF-β release from the polymers and its bio-regulating function in vivo. QCM result shows that PU bearing HSNGLPL peptide has affinity binding ability to TGF-β in vitro. Intramuscular implanting experiment further supports the enrichment efficiency of TGF-β on PU polymers in vivo. The detecting data involving intramuscular inflammatory infiltration triggered by the implants, myofiber regeneration, muscular fibrosis degree, and activation of endoplasmic reticulum stress (ER stress), evidence TGF-β can be released from PU polymers, and exerts regulating effects on the material-induced inflammation. Thus, our present results suggest it is feasible to improve biocompatibility of PU polymers in vivo, by pre-bearing bioactive molecules on materials before the implanting.

Elastic polyurethane bearing pendant TGF-β1 affinity peptide for potential tissue engineering applications

• An elastic degradable polyurethane (PU) bearing pendent HSNGLPL peptide for TGF-β1 affinity binding mimics the extracellular matrix function to retain and release growth factors.

In vivo immuno-reactivity analysis of the porous three-dimensional chitosan/SiO2 and chitosan/SiO2/hydroxyapatite hybrids: IN VIVO IMMUNO-REACTIVITY ANALYSIS

Inorganic/organic hybrid silica-chitosan (CS) scaffolds have promising potential for bone defect repair, due to the controllable mechanical properties, degradation behavior, and scaffold morphology. However, the precise in vivo immuno-reactivity of silica-CS hybrids with various compositions is still poorly defined. In this study, we fabricated the three-dimensional (3D) interconnected porous chitosan-silica (CS/SiO2 ) and chitosan-silica-hydroxyapatite (CS/SiO2 /HA) hybrids, through sol-gel process and 3D plotting skill, followed by the naturally or freeze drying separately. Scanning electron microscopy demonstrated the hybrids possessed the uniform geometric structure, while, transmission electron microscopy displayed nanoscale silica, or HA nanoparticles dispersed homogeneously in the CS matrix, or CS/silica hybrids. After intramuscular implantation, CS/SiO2 and CS/SiO2 /HA hybrids triggered a local and limited monocyte/macrophage infiltration and myofiber degeneration. Naturally dried CS/SiO2 hybrid provoked a more severe inflammation than the freeze-dried ones. Dendritic cells were attracted to invade into the implants embedded-muscle, but not be activated to prime the adaptive immunity, because the absence of cytotoxic T cells and B cells in muscle received the implants. Fluorescence-activated cell sorting (FACS) analysis indicated the implanted hybrids were incapable to initiate splenocytes activation. Plasma complement C3 enzyme linked immunosorbent assay (ELISA) assay showed the hybrids induced C3 levels increase in early implanting phase, and the subsequent striking decrease. Thus, the present results suggest that, in vivo, 3D plotted porous CS/SiO2 and CS/SiO2 /HA hybrids are relatively biocompatible in vivo, which initiate a localized inflammatory procedure, instead of a systematic immune response.

Calcium/Calmodulin-Dependent Protein Kinase IV (CaMKIV) Mediates Acute Skeletal Muscle Inflammatory Response

The objective of this study is to investigate the role of Calmodulin-dependent protein kinase IV (CaMKIV) in Cardiotoxin (CTX)-induced mice muscle inflammation. CTX injection i.m. was performed to induce B6 mice acute tibialis anterior (TA) muscle injury. The mice were then injected i.p. with the recombinant CaMKIV protein or its antagonist KN-93. Immunoblotting was used to assess Calmodulin (CaM) and CaMKIV levels. Immunofluorescence was used to detect intramuscular infiltration or major histocompatibility complex (MHC)-I expression in damaged muscle. The extent of infiltration was evaluated by fluorescent intensity analysis. Cytokines/chemokines levels were determined by qPCR. CaMKIV gene knockdown in C2C12 cells was performed in order to evaluate the effects of CaMKIV on immuno-behavior of muscle cells. CTX administration induced a strong up-regulation of CaM and p-CaMKIV levels in infiltrated mononuclear cells and regenerated myofibers. In vivo adding of the recombinant CaMKIV protein enhanced intramuscular infiltration of monocytes/macrophages in damaged muscle and increased the number of proinflammatory Ly-6C+F4/80+ macrophage cells. CaMKIV protein treatment induced a striking up-regulation of mRNA levels of IL-1, IL-6, MCP-1, and MCP-3 in CD45+ cells sorted from damaged muscle; increased the infiltration of CD8+ T cells; and induced the up-regulation of MHC-I in partial regenerated myofibers, which was rarely observed in muscle damage alone. Additionally, CaMKIV protein treatment diminished the regulatory T cells (Tregs) number and led to the damaged TA muscle repair delay. In vitro CaMKIV gene knockdown reversed IFN-γ-induced up-regulation of MHC-I/II and TLR3 in the differentiated C2C12 myotubes. CaMKIV can act as an immunostimulation molecule and enhances the acute muscle inflammatory responses.

Blocking of matrix metalloproteinases-13 responsive peptide in poly(urethane urea) for potential cartilage tissue engineering applications:

The matching of scaffold degradation rate with neotissue growth is required for tissue engineering applications. Timely provision of proper spaces especially for cartilage tissue engineering plays a pivotal role in chondrocyte cluster formation. In this study, poly(urethane urea) was synthesized using conventional two-stage method by extending the isocyanate group terminated prepolymers with different amounts of GPLGLWARK peptide, which responses the degrading induced by matrix metalloproteinase 13, the main proteinase for cartilage matrix degradation. The Fourier transform infrared spectrometer with the attenuated total reflection and 1H nuclear magnetic resonance spectra revealed that the peptides were introduced to poly(urethane urea) according to the characteristic absorption bands of the peptide and the newly formed urea bonds. The ultraviolet–visible spectroscopy spectra showed that the weight percentages of the peptide in the three poly(urethane urea) were 25%, 32%, and 35%. Atomic force microscopy images revealed that phase separation occurred in all poly(urethane urea) samples and became increasingly apparent with increasing amount of peptides introduced. Mechanical tests showed that the poly(urethane urea) strength increased with increasing amount of peptides in poly(urethane urea). Poly(urethane urea) proteolysis in matrix metalloproteinase 13 solution was more rapid than hydrolysis in aqueous buffer, and proteolysis rate was dependent on the amount of peptides in poly(urethane urea). Cell proliferation on the material surface in vitro displayed nontoxicity for all synthesized poly(urethane urea). In vivo subcutaneous implantation evaluation revealed the presence of local foreign body reactions triggered by poly(urethane urea) but was not due to peptide in poly(urethane urea). Moreover, the synthesized poly(urethane urea) with significant phase separation did not degrade under the matrix metalloproteinase 13 free subcutaneous environment, but poly(urethane urea) with minimal phase separation was degraded by attacking of the enzymes adsorbed on the hydrophobic surface through non-specific adsorption.

Anchoring TGF-β1 on biomaterial surface via affinitive interactions: Effects on spatial structures and bioactivity

Protein adsorption on biomaterial surfaces is clinically applied to increase therapeutic effects; however, this adsorption is possibly accompanied by conformational changes and results in loss of protein bioactivity or adverse reactions. In this research, a transforming growth factor β1 (TGF-β1) affinitive peptide HSNGLPL was grafted onto biopolymer surface to stabilize TGF-β1 spatial conformation after adhesion. The peptide with azide end group was combined with the propynyl pendant group on polyurethane via copper-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction. The final polymer was characterized by Fourier transform infrared spectroscopy and proton nuclear magnetic resonance spectroscopy, which indicated that the affinitive peptide was introduced to the polymer. Quartz crystal microbalance with dissipation (QCM-D) was performed to monitor TGF-β1 adsorption and desorption on the surfaces coated with polyurethane with and without peptide combination. Results showed that TGF-β1 adhered on polyurethane surface and formed a compact and rigid layer. This layer showed spatial structural change but presented a loose and diffuse layer on the peptide-grafted polyurethane surface, indicating stable spatial conformation after adherence. Similar regulations were observed on the two surfaces where BSA layer was coated in advance. In vivo animal experiments revealed that immune reactions and tissue regenerations occurred earlier on peptide-modified polyurethane than on polyurethane, which did not undergo peptide grafting. This finding confirmed that affinitive interactions may preserve TGF-β1 bioactivity on polymer surface after adsorption.

Calcium/Calmodulin-Dependent Protein Kinase IV Mediates IFN-γ-Induced Immune Behaviors in Skeletal Muscle Cells

Background/Aims: Whether calcium/calmodulin-dependent protein kinase IV (CaMKIV) plays a role in regulating immunologic features of muscle cells in inflammatory environment, as it does for immune cells, remains mostly unknown. In this study, we investigated the influence of endogenous CaMKIV on the immunological characteristics of myoblasts and myotubes received IFN-γ stimulation. Methods: C2C12 and murine myogenic precursor cells (MPCs) were cultured and differentiated in vitro, in the presence of pro-inflammatory IFN-γ. CaMKIV shRNA lentivirus transfection was performed to knockdown CaMKIV gene in C2C12 cells. pEGFP-N1-CaMKIV plasmid was delivered into knockout cells for recovering intracellular CaMKIV gene level. CREB1 antagonist KG-501 was used to block CREB signal. qPCR, immunoblot analysis, or immunofluorescence was used to detect mRNA and protein levels of CaMKIV, immuno-molecules, or pro-inflammatory cytokines and chemokines. Co-stimulatory molecules expression was assessed by FACS analysis. Results: IFN-γ induces the expression or up-regulation of MHC-I/II and TLR3, and the up-regulation of CaMKIV level in muscle cells. In contrast, CaMKIV knockdown in myoblasts and myotubes leads to expression inhibition of the above immuno-molecules. As well, CaMKIV knockdown selectively inhibits pro-inflammatory cytokines/chemokines, and co-stimulatory molecules expression in IFN-γ treated myoblasts and myotubes. Finally, CaMKIV knockdown abolishes IFN-γ induced CREB pathway molecules accumulation in differentiated myotubes. Conclusions: CaMKIV can be induced to up-regulate in muscle cells under inflammatory condition, and positively mediates intrinsic immune behaviors of muscle cells triggered by IFN-γ.