Phosphate burden induces vascular calcification through a NLRP3-caspase-1-mediated pyroptotic pathway.

AIMS: The aim of this study is to clarify the role of NLRP3 inflammasome in phosphate burden-induced vascular smooth muscle cell (VSMC) calcification. MAIN METHODS: VSMC calcification was induced using a high concentration of inorganic phosphate. After pharmacological inhibition or genetic silencing of the NLRP3 inflammasome, pyroptosis, or potassium efflux, the cells were examined by RT-qPCR, immunofluorescence, and western blotting to identify the NLRP3-mediated pathway for VSMC calcification. KEY FINDINGS: Calcified VSMCs with α-smooth muscle actin (α-SMA) disarray presented features of pyroptosis, including caspase-1 maturation, cleaved gasdermin D (GSDMD), and a high supernatant level of lactate dehydrogenase A. Pharmacological inhibitions of caspase-1 and pyroptosis attenuated VSMC calcification, whereas interleukin-1ß receptor antagonism did not. Unlike canonical NLRP3 activation, osteogenic VSMCs did not upregulate NLRP3 expression. However, NLRP3 genetic silencing or inhibitions, which targets different domains of the NLRP3 protein, could ameliorate VSMC calcification by aborting caspase-1 and GSDMD activation. Furthermore, potassium efflux through the inward-rectifier potassium channel, and not through the P2X7 receptor, triggered NLRP3 inflammasome activation and VSMC calcification. SIGNIFICANCE: In the present study, we identified a potassium efflux-triggered NLRP3-caspase-1-mediated pyroptotic pathway for VSMC calcification that is unique and different from the canonical NLRP3 inflammasome activation. Therefore, targeting this pathway may serve as a novel therapeutic strategy for vascular calcification.

Interaction of transcription factors Islet2 and Nr2f1b to control vascular patterning during zebrafish development.

Many regulators controlling arterial identity are well described; however, transcription factors that promote vein identity and vascular patterning have remained largely unknown. We previously identified the transcription factors Islet2 (Isl2) and Nr2f1b required for specification of the vein and tip cell identity mediated by notch pathway in zebrafish. However, the interaction between Isl2 and Nr2f1b is not known. In this study, we report that Nr2f2 plays minor roles on vein and intersegmental vessels (ISV) growth and dissect the genetic interactions among the three transcription factors Isl2, Nr2f1b, and Nr2f2 using a combinatorial knockdown strategy. The double knockdown of isl2/nr2f1b, isl2/nr2f2, and nr2f1b/nr2f2 showed the enhanced defects in vasculature including less completed ISV, reduced veins, and ISV cells. We further tested the genetic relationship among these three transcription factors. We found isl2 can regulate the expression of nr2f1b and nr2f2, suggesting a model where Isl2 functions upstream of Nr2f1b and Nr2f2. We hypothsized that Isl2 and Nr2f1b can function together through cis-regulatory binding motifs. In-vitro luciferase assay results, we showed that Isl2 and Nr2f1b can cooperatively enhance gene expression. Moreover, co-immunoprecipitation results indicated that Isl2 and Nr2f1b interact physically. Together, we showed that the interaction of the Nr2f1b and Nr2f2 transcription factors in combination with the Islet2 play coordinated roles in the vascular development of zebrafish.

Indoxyl Sulfate Mediates the Low Inducibility of the NLRP3 Inflammasome in Hemodialysis Patients.

The NLRP3 inflammasome is responsible for the maturation of caspase-1 and interleukin-1ß (IL-1ß). Despite the study about basal activity of the NLRP3 inflammasome in hemodialysis (HD) patients, little is known about its inducibility in the milieu of uremia. Peripheral blood mononuclear cells (PBMCs) isolated from 11 HD patients and 14 volunteers without a history of chronic kidney disease, as well as macrophages with or without the uremic toxin indoxyl sulfate (IS) pretreatment, underwent canonical NLRP3 inflammasome induction. Despite the high plasma levels of IL-1ß in HD patients, caspase-1 and IL-1ß in the PBMCs of HD patients remained predominantly immature and were not secreted in response to the canonical stimulus. In addition, while IS alone facilitated the inflammasome-independent secretion of IL-1ß from macrophages, IS exposure before induction reduced the inducibility of the NLRP3 inflammasome, characterized by insufficient maturation of caspase-1. The low expression of inflammasome components, which was observed in both IS-pretreated cells and the PBMCs of HD patients, was probably responsible for the low inducibility.

Characterization of the monoclonal antibody specific to the ORF72 protein of koi herpesvirus and cellular distribution analysis of the viral protein.

Koi herpesvirus (KHV) is an emerging pathogen of koi and common carp that causes a severe disease and mass mortality of infected fish. The KHV ORF72 protein is an important capsid protein that has been suggested to be a candidate for the development of diagnostic reagents and KHV vaccines. The purpose of this study was to clone and express the KHV ORF72 gene for further preparation of a specific monoclonal antibody (mAb) and to analyse cellular distribution of the viral protein. The mAb 3E1 could specifically recognize the expressed ORF72 protein of transfected cells by indirect immunofluorescence, and the antigenic site recognized by the mAb 3E1 was mapped to the region of N-terminal 124 residues of KHV ORF72. This mAb was further demonstrated to specifically detect the KHV-infected fish tissue by immunohistochemistry, thereby suggesting its high diagnostic potential. In addition, the cellular distribution analysis of the KHV ORF72 protein revealed that the region of amino acid residues 125-247 was related to mitochondrial localization and proliferation. Furthermore, a putative nuclear export signal (NES) of ORF72 at the residues 201-212 was confirmed on the basis of its function associated with NES activity.

Fine-tune regulation of carboxypeptidase N1 controls vascular patterning during zebrafish development.

Vascular development is regulated by complicated signals and molecules in vertebrates. In this study, we characterized a novel function of carboxypeptidase N1 (Cpn1) in the vasculature. We show that cpn1 mRNA is expressed in developing vessels. The knockdown of cpn1 by morpholino injection impairs the growth of intersegmental vessels (ISV) and caudal vein plexus (CVP), suggesting the role of cpn1 in vascular development. We showed that vascular defects are not caused by cell death but are due to the impairment of migration and proliferation. Consistent with vascular growth defects, loss of cpn1 affects the expression of the vascular markers flt4, mrc1, flk, stabilin, and ephrinb2. Furthermore, the overexpression of cpn1 impaired the growth of ISV and CVP, but the remodeling expression of vascular markers was different from the knockdown of cpn1, indicating the differential regulation mechanisms in cpn1-overexpressing embryos. We examine the interaction between cpn1 and multiple signals and observed that cpn1 is regulated by Notch/VEGF signals for ISV growth and likely regulates BMP signals for CVP patterning. In conclusion, we demonstrate that cpn1 has a critical role in the vascular development of zebrafish. We also reveal a fine-tune regulation of cpn1 that controls vascular patterning mediated by multiple signals.

Low power consumption and high-contrast light scattering based on polymer-dispersed liquid crystals doped with silver-coated polystyrene microspheres.

Polymer-dispersed liquid crystals (PDLCs) have attracted considerable attention for optical device applications in recent years. However, the high operating voltage of PDLCs limits their applications. This study reports a simple approach used for the first time to decrease the operating voltage of PDLCs by means of doping 3 µm-diameter silver-coated polystyrene microspheres (Ag-coated PSMSs) into PDLCs. Ag-coated PSMSs construct an induced electric field between each other when an external electric field is applied. This induced electric field can enhance the effective electric field so the operating voltage can be actively reduced from 77 V to 40 V. Such PDLCs also possess a high contrast ratio of >50 and a high on-state transmittance of ~73%. Therefore, PDLCs doped with Ag-coated PSMSs maintain a high contrast ratio and improve their electro-optical properties.

Nr2f1b control venous specification and angiogenic patterning during zebrafish vascular development.

BACKGROUND: The specification of vein and the patterning of intersegmental vessels (ISV) controlled by transcription factor is not fully characterized. The orphan nuclear receptor Chicken ovalbumin upstream promoter transcription factor II (CoupTFII, a.k.a NR2F2) positively regulates vein identity in mice. In this study, we show that nr2f1b is important for vein and tip cell identity during zebrafish development. RESULTS: Nr2f1b mRNA is expressed in ventral lateral mesoderm at 15S stage and in vessels at 24 hpf consistent with a role in early vascular specification. Morpholino knockdown of nr2f1b results in a decrease in both vein cell number and expression of the vein specific marker flt4 and mrc1, suggested its role in venous specification. We also show loss of nr2f1b reduced ISV cell number and impairs ISV growth, which is likely due to the impairment of angiogenic cells migration and/or proliferation by time-lapse imaging. Consequently, nr2f1b morphants showed pericardial edema and circulation defects. Overexpression of nr2f1b under the fli promoter increases the number of venous cells and ISV endothelial cells indicated the function of nr2f1b is required and necessary for vascular development. We further showed that nr2f1b likely interact with Notch signalling. nr2f1b expression is increased in rbpsuh morphants and DAPT-treatment embryos suggested nr2f1b is negatively regulated by Notch activity. CONCLUSIONS: We show nr2f1b control venous specification and angiogenic patterning during zebrafish vascular development, which is mediated by Notch signalings.