Loss of Smooth Muscle Tenascin-X Inhibits Vascular Remodeling Through Increased TGF-ß Signaling.

BACKGROUND: Vascular smooth muscle cells (VSMCs) are highly plastic. Vessel injury induces a phenotypic transformation from differentiated to dedifferentiated VSMCs, which involves reduced expression of contractile proteins and increased production of extracellular matrix and inflammatory cytokines. This transition plays an important role in several cardiovascular diseases such as atherosclerosis, hypertension, and aortic aneurysm. TGF-ß (transforming growth factor-ß) is critical for VSMC differentiation and to counterbalance the effect of dedifferentiating factors. However, the mechanisms controlling TGF-ß activity and VSMC phenotypic regulation under in vivo conditions are poorly understood. The extracellular matrix protein TN-X (tenascin-X) has recently been shown to bind TGF-ß and to prevent it from activating its receptor. METHODS: We studied the role of TN-X in VSMCs in various murine disease models using tamoxifen-inducible SMC-specific knockout and adeno-associated virus-mediated knockdown. RESULTS: In hypertensive and high-fat diet-fed mice, after carotid artery ligation as well as in human aneurysmal aortae, expression of Tnxb, the gene encoding TN-X, was increased in VSMCs. Mice with smooth muscle cell-specific loss of TN-X (SMC-Tnxb-KO) showed increased TGF-ß signaling in VSMCs, as well as upregulated expression of VSMC differentiation marker genes during vascular remodeling compared with controls. SMC-specific TN-X deficiency decreased neointima formation after carotid artery ligation and reduced vessel wall thickening during Ang II (angiotensin II)-induced hypertension. SMC-Tnxb-KO mice lacking ApoE showed reduced atherosclerosis and Ang II-induced aneurysm formation under high-fat diet. Adeno-associated virus-mediated SMC-specific expression of short hairpin RNA against Tnxb showed similar beneficial effects. Treatment with an anti-TGF-ß antibody or additional SMC-specific loss of the TGF-ß receptor reverted the effects of SMC-specific TN-X deficiency. CONCLUSIONS: In summary, TN-X critically regulates VSMC plasticity during vascular injury by inhibiting TGF-ß signaling. Our data indicate that inhibition of vascular smooth muscle TN-X may represent a strategy to prevent and treat pathological vascular remodeling.

Tenascin-X Mediates Flow-Induced Suppression of EndMT and Atherosclerosis.

BACKGROUND: Endothelial-to-mesenchymal transition (EndMT) has been identified as a critical driver of vascular inflammation and atherosclerosis, and TGF-ß (transforming growth factor ß) is a key mediator of EndMT. Both EndMT and atherosclerosis are promoted by disturbed flow, whereas unidirectional laminar flow limits EndMT and is atheroprotective. How EndMT and endothelial TGF-ß signaling are regulated by different flow patterns is, however, still poorly understood. METHODS: Flow chamber experiments in vitro and endothelium-specific knockout mice were used to study the role of tenascin-X in the regulation of EndMT and atherosclerosis as well as the underlying mechanisms. RESULTS: In human endothelial cells as well as in human and mouse aortae, unidirectional laminar flow but not disturbed flow strongly increased endothelial expression of the extracellular matrix protein TN-X (tenascin-X) in a KLF4 (Krüppel-like factor 4) dependent manner. Mice with endothelium-specific loss of TN-X (EC-Tnxb-KO) showed increased endothelial TGF-ß signaling as well as increased endothelial expression of EndMT and inflammatory marker genes. When EC-Tnxb-KO mice were subjected to partial carotid artery ligation, we observed increased vascular remodeling. EC-Tnxb-KO mice crossed to low-density lipoprotein receptor-deficient mice showed advanced atherosclerotic lesions after being fed a high-fat diet. Treatment of EC-Tnxb-KO mice with an anti-TGF-beta antibody or additional endothelial loss of TGF-beta receptors 1 and 2 normalized endothelial TGF-beta signaling and prevented EndMT. In in vitro studies, we found that TN-X through its fibrinogen-like domain directly interacts with TGF-ß and thereby interferes with its binding to the TGF-ß receptor. CONCLUSIONS: In summary, we show that TN-X is a central mediator of flow-induced inhibition of EndMT, endothelial inflammation and atherogenesis, which functions by binding to and by blocking the activity of TGF-ß. Our data identify a novel mechanism of flow-dependent regulation of vascular TGF-ß, which holds promise for generating new strategies to prevent vascular inflammation and atherosclerosis.

ClC-3 promotes angiotensin II-induced reactive oxygen species production in endothelial cells by facilitating Nox2 NADPH oxidase complex formation.

Recent evidence suggests that ClC-3, a member of the ClC family of Cl- channels or Cl-/H+ antiporters, plays a critical role in NADPH oxidase-derived reactive oxygen species (ROS) generation. However, the underling mechanisms remain unclear. In this study we investigated the effects and mechanisms of ClC-3 on NADPH oxidase activation and ROS generation in endothelial cells. Treatment with angiotensin II (Ang II, 1 µmol/L) significantly elevated ClC-3 expression in cultured human umbilical vein endothelial cells (HUVECs). Furthermore, Ang II treatment increased ROS production and NADPH oxidase activity, an effect that could be significantly inhibited by knockdown of ClC-3, and further enhanced by overexpression of ClC-3. SA-ß-galactosidase staining showed that ClC-3 silencing abolished Ang II-induced HUVEC senescence, whereas ClC-3 overexpression caused the opposite effects. We further showed that Ang II treatment increased the translocation of p47phox and p67phox from the cytosol to membrane, accompanied by elevated Nox2 and p22phox expression, which was significantly attenuated by knockdown of ClC-3 and potentiated by overexpression of ClC-3. Moreover, overexpression of ClC-3 increased Ang II-induced phosphorylation of p47phox and p38 MAPK in HUVECs. Pretreatment with a p38 inhibitor SB203580 abolished ClC-3 overexpression-induced increase in p47phox phosphorylation, as well as NADPH oxidase activity and ROS generation. Our results demonstrate that ClC-3 acts as a positive regulator of Ang II-induced NADPH oxidase activation and ROS production in endothelial cells, possibly via promoting both Nox2/p22phox expression and p38 MAPK-dependent p47phox/p67phox membrane translocation, then increasing Nox2 NADPH oxidase complex formation.

[Research on Pyrolysis Process of Kevlar Fibers with Thermogravimetric Analysis coupled and Fourier Transform Infrared Spectroscopy].

Modern industrial application and technologies require materials with superior mechanical and thermal properties. Kevlar fibers have been known as fibrous materials with good properties of high strength and high decomposition temperature which have become a hot research field in recent years. The properties of fibrous materials depend on their structures and compositions. Thermal decomposition processing of the materials is of great significance for their structures and thermal properties. As a new technique, thermogravimetric (TG) analysis coupled with Fourier transform infrared spectroscopy(FTIR), are able to analyze materials not only qualitatively but also quantitatively. This method has obvious advantages in researching the thermal decomposition of many materials. However, the thermal decomposition processing of Kevlar fibers is rarely reported in the literature, therefore, we firstly studied the pyrolysis behavior of Kevlar fibers with thermogravimetric analysis coupled with Fourier transform infrared spectroscopy at the temperature of 30~800 ℃. We not only obtained the processing of the Kevlar fibers' thermal decomposition with great details but also the products of every stage. Experimental results exhibited that the decomposition of Kevlar fibers has experienced three stages： 100~240, 240~420 and 420~800 ℃. The weight loss of Kevlar fibers was quite slow before 500 ℃. The third stage was the main stage of the decomposition, and the amount of residue finally reached to a mass percent of 56.21%. FTIR analysis illustrated that free water released from Kevlar fibers at the first stage, followed by the dehydration and depolymerization which made polymer chains short. Finally the fiber fragments further reacted and produced the gases of small molecular mass, and the main products were water, ammonia, carbon monoxide and carbon dioxide. Generation rate of water was increased; the emission of ammonia was at the same rate; carbon monoxide was only produced at the temperature of 515~630 ℃, then turned into carbon dioxide. The release of carbon dioxide was on rise because of the conversion process of carbon monoxide, and then dropped to a certain value.

Clustered Regularly Interspaced Short Palindromic Repeats and Clustered Regularly Interspaced Short Palindromic Repeats-Associated Protein 9 System: Factors Affecting Precision Gene Editing Efficiency and Optimization Strategies.

The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) system is a powerful genomic DNA editing tool. The increased applications of gene editing tools, including the CRISPR-Cas system, have contributed to recent advances in biological fields, such as genetic disease therapy, disease-associated gene screening and detection, and cancer therapy. However, the major limiting factor for the wide application of gene editing tools is gene editing efficiency. This review summarizes the recent advances in factors affecting the gene editing efficiency of the CRISPR-Cas9 system and the CRISPR-Cas9 system optimization strategies. The homology-directed repair efficiency-related signal pathways and the form and delivery method of the CRISPR-Cas9 system are the major factors that influence the repair efficiency of gene editing tools. Based on these influencing factors, several strategies have been developed to improve the repair efficiency of gene editing tools. This review provides novel insights for improving the repair efficiency of the CRISPR-Cas9 gene editing system, which may enable the development and improvement of gene editing tools.

Changes in the TCR repertoire of T-cell subsets during culture of cytokine-induced killer cells.

Cytokine-induced killer (CIK) cells are a group of highly heterogeneous T cells expressing diverse T-cell antigen receptors (TCRs), but possessing wide major histocompatibility complex (MHC)-unrestricted antitumour activity, which have been used in the treatment of many tumours. However, the clonal diversity of different T-cell subsets and whether there is a certain clonal preference during CIK culture remains to be clarified. We found that the proportion of oligoclones seen in some subfamilies of freshly isolated T cells decreased in varying degrees after culturing. This indicates that the diversity of T-cell clones had been restored to a certain extent. The diversity differed among different T-cell subsets, as some TCR subfamily oligoclones mainly appeared in CD3+ CD56+ cells, which also indicates the heterogeneity of the CIK cell composition.

Involvement of MicroRNA-27a-3p in the Licorice-Induced Alteration of Cd28 Expression in Mice.

Licorice has previously been shown to affect gene expression in cells; however, the underlying mechanisms remain to be clarified. We analyzed the microRNA expression profile of serum from mice treated by gavage with licorice decoction, and obtained 11 differentially expressed microRNAs (DEmiRNAs). We also screened differentially expressed genes (DEgenes) based on RNA-Seq data, and 271 common genes were identified by intersection analysis of the predicted target genes of 11 DEmiRNAs and the DEgenes. The miRNA-gene network showed that most of the hub genes were immune-related. KEGG enrichment analysis of the 271 genes identified three significant pathways, and the 21 genes involved in these three pathways, and the 11 DEmiRNAs, were constructed into a miRNA pathway-target gene network, in which mmu-miR-27a-3p stood out. Compared to ImmPort, there were 13 immune genes within the above group of 21 genes, and three intersected with the mmu-miR-27a-3p predicted target genes, Cd28, Grap2 and Cxcl12, of which the expression of Cd28 changed most significantly. We confirmed the regulation of Cd28 by mmu-miR-27a-3p using a dual-luciferase assay, and further confirmed that overexpression of mmu-miR-27a-3p could significantly downregulate the expression of Cd28 in lymphocytes. These results indicate that mmu-miR-27a-3p could be involved in the licorice-mediated regulation of the expression of Cd28 in mice.

Protein kinase N2 mediates flow-induced endothelial NOS activation and vascular tone regulation.

Formation of NO by endothelial NOS (eNOS) is a central process in the homeostatic regulation of vascular functions including blood pressure regulation, and fluid shear stress exerted by the flowing blood is a main stimulus of eNOS activity. Previous work has identified several mechanosensing and -transducing processes in endothelial cells, which mediate this process and induce the stimulation of eNOS activity through phosphorylation of the enzyme via various kinases including AKT. How the initial mechanosensing and signaling processes are linked to eNOS phosphorylation is unclear. In human endothelial cells, we demonstrated that protein kinase N2 (PKN2), which is activated by flow through the mechanosensitive cation channel Piezo1 and Gq/G11-mediated signaling, as well as by Ca2+ and phosphoinositide-dependent protein kinase 1 (PDK1), plays a pivotal role in this process. Active PKN2 promoted the phosphorylation of human eNOS at serine 1177 and at a newly identified site, serine 1179. These phosphorylation events additively led to increased eNOS activity. PKN2-mediated eNOS phosphorylation at serine 1177 involved the phosphorylation of AKT synergistically with mTORC2-mediated AKT phosphorylation, whereas active PKN2 directly phosphorylated human eNOS at serine 1179. Mice with induced endothelium-specific deficiency of PKN2 showed strongly reduced flow-induced vasodilation and developed arterial hypertension accompanied by reduced eNOS activation. These results uncover a central mechanism that couples upstream mechanosignaling processes in endothelial cells to the regulation of eNOS-mediated NO formation, vascular tone, and blood pressure.

Disturbed flow-induced Gs-mediated signaling protects against endothelial inflammation and atherosclerosis.

Atherosclerosis develops preferentially in areas of the arterial system, in which blood flow is disturbed. Exposure of endothelial cells to disturbed flow has been shown to induce inflammatory signaling, including NF-κB activation, which leads to the expression of leukocyte adhesion molecules and chemokines. Here, we show that disturbed flow promotes the release of adrenomedullin from endothelial cells, which in turn activates its Gs-coupled receptor calcitonin receptor-like receptor (CALCRL). This induces antiinflammatory signaling through cAMP and PKA, and it results in reduced endothelial inflammation in vitro and in vivo. Suppression of endothelial expression of Gαs, the α subunit of the G-protein Gs; CALCRL; or adrenomedullin leads to increased disturbed flow-induced inflammatory signaling in vitro and in vivo. Furthermore, mice with induced endothelial-specific deficiency of Gαs, CALCRL, or adrenomedullin show increased atherosclerotic lesions. Our data identify an antiinflammatory signaling pathway in endothelial cells stimulated by disturbed flow and suggest activation of the endothelial adrenomedullin/CALCRL/Gs system as a promising approach to inhibit progression of atherosclerosis.

Construction of nickel-doped cobalt hydroxides hexagonal nanoplates for advanced oxygen evolution electrocatalysis.

Oxygen vacancy of the catalyst is one of the most significant factors affecting the oxygen evolution reaction (OER) electrocatalysis, a key process for overall water splitting. Here, we report the oxygen vacancy engineering of Co(OH)2 hexagonal nanoplates by doping Ni element, which can efficiently lower the average Co oxidation state and thus optimize the Gibbs free energy of the intermediates, giving rise to the largely promoted OER performances. Impressively, the as-obtained oxygen-vacancy-rich Ni-doped Co(OH)2 hexagonal nanoplates manifest OER overpotential as low as 238 mV at 10 mA cm-2 in 1 M KOH solution, being superior to most of binary CoNi hydroxides. In accordance with the high OER activity, it also displays excellent durability in strong alkaline media over 40 h, presenting an advanced electrocatalyst for OER.

Fabrication of In Situ Nanofiber-Reinforced Molecular Composites by Nonequilibrium Self-Assembly.

Although the concept of molecular composites (MCs) is very promising, there are major obstacles arising from the immiscibility of the rigid-rod with the random-coil polymers. Here, we developed a novel method for fabricating an in situ reinforced MC system with nonequilibrium self-assembled nanofibrous structures based on bisphenol A epoxy resin, 4,4'-diaminodiphenylsulfone, bismaleimide, and a polyphenylene ether (PPO) oligomer. A variety of spectroscopic and morphological techniques were used to probe the structural evolution from the emergence of nanofibrils, to growth and aggregation of nanofibers, and then to the formation of in situ reinforced MC with strong interfacial interactions. The in situ nanofibers within the polymer matrix could be formed by the polymerization force extruding the PPO phase through the interspaces within the simultaneous interpenetrating network polymers during the cure process of the thermosetting resin system. Compared to the control sample, the in situ nanofiber-reinforced MC exhibited better thermal properties and flame retardancy. In particular, the obtained MC showed a significant improvement in glass transition temperature and mechanical properties, which were mainly attributed to the restriction of high thermal stability of PPO on the segmental motion of polymer chains, the toughening and reinforcement behaviors of PPO nanofibers on the matrix, and the chemical interaction at the PPO/matrix interface.

A study on the protection to relics and the related problems with diffuse reflectance spectroscopy.

The application of diffuse reflectance spectroscopy to relic protection is studied by using a self-made fiber optics reflectance spectrophotometer. The major work done includes: (1) the composition of pigment on colored relics and its changes are identified; (2) the change on metal surface is monitored; (3) the reflectance spectrum characteristics of relic protection materials are studied. The results tell that diffuse reflectance spectroscopy is a new protection technique, characterized by its quickness and non-destructiveness to the relic.

[Studies on relics conservation by diffuse reflectance spectroscopy].

A diffuse reflectance spectroscopic method was developed to investigate relics conservation with a self-made fiber optics reflectance spectrophotometer. With this technique (1) identifying pigments on the color relics was done by comparing the shapes of reflectance spectra and the positions of reflectance peaks or first derivative peaks with those of related standard pigments; (2) changes of surface states of metals were monitored. From the investigation on the reflectance spectra of copper and its rust product, we can easily get some information about the metal surface, so diffuse reflectance spectroscopy shows special function in the research on metal surface; (3) the diffuse reflectance spectroscopic characteristics of the materials used in protection and restoration of cultural heritages were studied, which can help us choose proper protecting and restoring materials. From our studies it was verified that diffuse reflectance spectroscopy is a quick, simple and non-destructive analytical technique. The study makes diffuse reflectance spectroscopy a new means for relic protection, and opens a new application area for diffuse reflectance spectroscopy as well.

Multifunctional cyclotriphosphazene/hexagonal boron nitride hybrids and their flame retarding bismaleimide resins with high thermal conductivity and thermal stability.

A novel hybridized multifunctional filler (CPBN), cyclotriphosphazene/hexagonal boron nitride (hBN) hybrid, was synthesized by chemically coating hBN with hexachlorocyclotriphosphazene and p-phenylenediamine, its structure was systemically characterized. Besides, CPBN was used to develop new flame retarding bismaleimide/o,o'-diallylbisphenol A (BD) resins with simultaneously high thermal conductivity and thermal stability. The nature of CPBN has a strong influence on the flame behavior of the composites. With the addition of only 5 wt % CPBN to BD resin, the thermal conductivity increases 2 times; meanwhile the flame retardancy of BD resin is remarkably increased, reflected by the increased limited oxygen index, much longer time to ignition, significantly reduced heat release rate. The thermogravimetric kinetics, structures of chars and pyrolysis gases, and cone calorimeter tests were investigated to reveal the unique flame retarding mechanism of CPBN/BD composites. CPBN provides multieffects on improving the flame retardancy, especially in forming a protective char layer, which means a more thermally stable and condensed barrier for heat and mass transfer, and thus protecting the resin from further combustion.

A nontoxic and low-cost hydrothermal route for synthesis of hierarchical Cu2ZnSnS4 particles.

We explore a facile and nontoxic hydrothermal route for synthesis of a Cu2ZnSnS4 nanocrystalline material by using l-cysteine as the sulfur source and ethylenediaminetetraacetic acid (EDTA) as the complexing agent. The effects of the amount of EDTA, the mole ratio of the three metal ions, and the hydrothermal temperature and time on the phase composition of the obtained product have been systematically investigated. The addition of EDTA and an excessive dose of ZnCl2 in the hydrothermal reaction system favor the generation of kesterite Cu2ZnSnS4. Pure kesterite Cu2ZnSnS4 has been synthesized at 180°C for 12 h from the reaction system containing 2 mmol of EDTA at 2:2:1 of Cu/Zn/Sn. It is confirmed by Raman spectroscopy that those binary and ternary phases are absent in the kesterite Cu2ZnSnS4 product. The kesterite Cu2ZnSnS4 material synthesized by the hydrothermal process consists of flower-like particles with 250 to 400 nm in size. It is revealed that the flower-like particles are assembled from single-crystal Cu2ZnSnS4 nanoflakes with ca. 20 nm in size. The band gap of the Cu2ZnSnS4 nanocrystalline material is estimated to be 1.55 eV. The films fabricated from the hierarchical Cu2ZnSnS4 particles exhibit fast photocurrent responses under intermittent visible-light irradiation, implying that they show potentials for use in solar cells and photocatalysis.

Modification of liposomes with proteins by dansyl-labeled heterobifunctional crosslinker.

The introduction of a fluorescent chromaphore into bifunctional crosslinkers results in a molecule with normal crosslinker properties and a fluorescent group for straightforward quantification. This work describes the synthesis of the dansyl-labeled heterobifunctional crosslinker N-succinimidyl ε-N-dansyl α-N-(acetylthio)acetyllysine (dansyl-ATA-lysine-NHS) containing reactive N-hydroxysuccinimidyl (NHS) ester and sulfhydryl groups. The application of this crosslinker to conjugation of bovine serum albumin (BSA) protein to the surface of a liposome containing maleimide functions is also demonstrated. BSA was modified with the dansyl-labeled crosslinker and subsequently conjugated to liposomes containing reactive phospholipid derivative N-[4-(p-maleimidophenyl)butyryl]phosphatidylethanolamine and the degree of modification and conjugation were quantitatively determined by measuring the fluorescence emission of the dansyl group. The reliability of the fluorescence quantification was confirmed by a micro bio-barcode assay protein assay.