Pediatric high-altitude pulmonary edema and acute mountain sickness: Clinical features and risk determinants.

OBJECTIVE: This investigation aimed to delineate the clinical manifestations associated with high-altitude pulmonary edema (HAPE) and acute mountain sickness (AMS) in pediatric populations and find the risk factors of HAPE. METHODS: We conducted a retrospective analysis of clinical data from children under 18 years diagnosed with HAPE and AMS at an average altitude of 3000 m. The clinical data between these two groups were compared. RESULTS: The study encompassed 74 pediatric patients, 27 with AMS and 47 with HAPE. HAPE presentations included classic HAPE (55.3%), reentry HAPE (27.7%), and high-altitude resident pulmonary edema (HARPE, 17.0%). Notably, 87.2% of HAPE cases were male, and 68.1% had a high body mass index (BMI). HARPE instances followed viral infections, prominently SARS-CoV-2. HAPE cases exhibited higher BMI, respiratory tract infections within 1 week preceding symptom onset, an increase in white blood cell counts (WBCs), lower peripheral arterial oxygen saturation (SpO2), and higher heart rate compared to the AMS group. Multivariate logistic regression pinpointed high BMI as an independent HAPE risk factor (odds ratio = 19.389, 95% confidence interval: 1.069-351.759, p = .045). CONCLUSION: HAPE occurs predominantly in males, with high BMI identified as a critical independent risk factor. The study underscores the need for heightened awareness and preventive strategies against HAPE in children at high altitudes.

scPML: pathway-based multi-view learning for cell type annotation from single-cell RNA-seq data.

Recent developments in single-cell technology have enabled the exploration of cellular heterogeneity at an unprecedented level, providing invaluable insights into various fields, including medicine and disease research. Cell type annotation is an essential step in its omics research. The mainstream approach is to utilize well-annotated single-cell data to supervised learning for cell type annotation of new singlecell data. However, existing methods lack good generalization and robustness in cell annotation tasks, partially due to difficulties in dealing with technical differences between datasets, as well as not considering the heterogeneous associations of genes in regulatory mechanism levels. Here, we propose the scPML model, which utilizes various gene signaling pathway data to partition the genetic features of cells, thus characterizing different interaction maps between cells. Extensive experiments demonstrate that scPML performs better in cell type annotation and detection of unknown cell types from different species, platforms, and tissues.

Correction: Mononuclear-macrophages but not neutrophils act as major infiltrating anti-leptospiral phagocytes during leptospirosis.

[This corrects the article DOI: 10.1371/journal.pone.0181014.].

Etiological Analysis of Viral Encephalitis in Children in Zhejiang Province from 2018 to 2019.

OBJECTIVE: To investigate the common pathogens of viral encephalitis (VE) in children, and to provide guidance for the empirical diagnosis and treatment of patients with VE. METHODS: A total of 227 cerebrospinal fluid (CSF) samples were collected from pediatric patients with VE in Zhejiang province from January 2018 to December 2019. The samples were tested using multiplex and singleplex Reverse Transcription-Polymerase Chain Reaction (RT-PCR) with primers specific to enterovirus (EV), varicella-zoster virus (VZV), mumps virus (MuV), cytomegalovirus (CMV), herpes simplex virus type 1 (HSV-1)/type 2 (HSV-2), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV-6). The data of the two analyses were compared and then verified using Sanger sequencing. RESULTS: Of the 227 CSF samples, 90 were shown to be positive for multiplex RT-PCR with a positivity rate of 39.65% and a 95% confidence interval (33.2%, 46.1%). EV was the most common cause of VE, followed by EBV, HHV-6, MuV, CMV, VZV, and HSV-1. Most included cases occurred in summer, accounting for 49.78% of all cases. For EV, EBV, and HSV-2, multiplex RT-PCR showed a positivity rate of 34.36%, which was not statistically different from that of 30.4% shown by singleplex RT-PCR. The sequences of EV, EBV, VZV, MuV, CMV, HSV-1, HHV-6, and HSV-2 were confirmed by sequencing the PCR products obtained from multiplex and singleplex PCR. CONCLUSIONS: In children, VE is more prevalent in the summer than in other seasons in Zhejiang province, and EV may be the most common causative pathogen.

Diagnostic Value of Interferon-Gamma Release Assays for Tuberculosis in the Immunocompromised Population.

Interferon-gamma release assays (IGRAs) are widely used in the diagnosis of Mycobacterium tuberculosis (M. tuberculosis) infection by detecting interferon-γ released by previously sensitized T-cells in-vitro. Currently, there are two assays based on either enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunospot (ELISPOT) technology, with several generations of products available. The diagnostic value of IGRAs in the immunocompromised population is significantly different from that in the immunocompetent population because their results are strongly affected by the host immune function. Both physiological and pathological factors can lead to an immunocompromised situation. We summarized the diagnostic value and clinical recommendations of IGRAs for different immunocompromised populations, including peoplewith physiological factors (pregnant and puerperal women, children, and older people), as well as people with pathological factors (solid organ transplantation recipients, combination with human immunodeficiency virus infection, diabetes mellitus, end-stage renal disease, end-stage liver disease, and chronic immune-mediated inflammatory diseases). Though the performance of IGRAs is not perfect and often requires a combination with other diagnostic strategies, it still has some value in the immunocompromised population. Hopefully, the newly developed IGRAs could better target this population.

M16-Type Metallopeptidases Are Involved in Virulence for Invasiveness and Diffusion of Leptospira interrogans and Transmission of Leptospirosis.

BACKGROUND: Leptospirosis is a global zoonotic infectious disease caused by Leptospira interrogans. The pathogen rapidly invades into hosts and diffuses from bloodstream into internal organs and excretes from urine to cause transmission of leptospirosis. However, the mechanism of leptospiral invasiveness remains poorly understood. METHODS: Proteolytic activity of M16-type metallopeptidases (Lep-MP1/2/3) of L. interrogans was determined by spectrophotometry. Expression and secretion of Lep-MP1/2/3 during infection of cells were detected by quantitative reverse-transcription polymerase chain reaction, Western blot assay, and confocal microscopy. Deletion and complementation mutants of the genes encoding Lep-MP1/2/3 were generated to determine the roles of Lep-MP1/2/3 in invasiveness using transwell assay and virulence in hamsters. RESULTS: Leptospira interrogans but not saprophytic Leptospira biflexa strains were detectable for Lep-MP-1/2/3-encoding genes. rLep-MP1/2/3 hydrolyzed extracellular matrix proteins, but rLep-MP1/3 displayed stronger proteolysis than rLep-MP2, with 123.179/340.136 µmol/L Km and 0.154/0.159 s-1 Kcat values. Expression, secretion and translocation of Lep-MP1/2/3 during infection of cells were increased. ΔMP1/3 but not ΔMP2 mutant presented attenuated transmigration through cell monolayers, decreased leptospiral loading in the blood, lungs, liver, kidneys, and urine, and 10/13-fold decreased 50% lethal dose and milder histopathologic injury in hamsters. CONCLUSIONS: Lep-MP1 and 3 are involved in virulence of L. interrogans in invasion into hosts and diffusion in vivo, and transmission of leptospirosis.

Endocytic recycling and vesicular transport systems mediate transcytosis of Leptospira interrogans across cell monolayer.

Many bacterial pathogens can cause septicemia and spread from the bloodstream into internal organs. During leptospirosis, individuals are infected by contact with Leptospira-containing animal urine-contaminated water. The spirochetes invade internal organs after septicemia to cause disease aggravation, but the mechanism of leptospiral excretion and spreading remains unknown. Here, we demonstrated that Leptospira interrogans entered human/mouse endothelial and epithelial cells and fibroblasts by caveolae/integrin-ß1-PI3K/FAK-mediated microfilament-dependent endocytosis to form Leptospira (Lep)-vesicles that did not fuse with lysosomes. Lep-vesicles recruited Rab5/Rab11 and Sec/Exo-SNARE proteins in endocytic recycling and vesicular transport systems for intracellular transport and release by SNARE-complex/FAK-mediated microfilament/microtubule-dependent exocytosis. Both intracellular leptospires and infected cells maintained their viability. Leptospiral propagation was only observed in mouse fibroblasts. Our study revealed that L. interrogans utilizes endocytic recycling and vesicular transport systems for transcytosis across endothelial or epithelial barrier in blood vessels or renal tubules, which contributes to spreading in vivo and transmission of leptospirosis.

vWA proteins of Leptospira interrogans induce hemorrhage in leptospirosis by competitive inhibition of vWF/GPIb-mediated platelet aggregation.

BACKGROUD: Leptospira interrogans is the major causative agent of leptospirosis, a worldwide zoonotic disease. Hemorrhage is a typical pathological feature of leptospirosis. Binding of von Willebrand factor (vWF) to platelet glycoprotein-Ibα (GPIbα) is a crucial step in initiation of platelet aggregation. The products of L. interrogans vwa-I and vwa-II genes contain vWF-A domains, but their ability to induce hemorrhage has not been determined. METHODS: Human (Hu)-platelet- and Hu-GPIbα-binding abilities of the recombinant proteins expressed by L. interrogans strain Lai vwa-I and vwa-II genes (rLep-vWA-I and rLep-vWA-II) were detected by flowcytometry, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Hu-platelet aggregation and its signaling kinases and active components were detected by lumiaggregometry, Western analysis, spectrophotometry and confocal microscopy. Hu-GPIbα-binding sites in rLep-vWA-I and rLep-vWA-II were identified by SPR/ITC measurements. FINDINGS: Both rLep-vWA-I and rLep-vWA-II were able to bind to Hu-platelets and inhibit rHu-vWF/ristocetin-induced Hu-platelet aggregation, but Hu-GPIbα-IgG, rLep-vWA-I-IgG and rLep-vWA-II-IgG blocked this binding or inhibition. SPR and ITC revealed a tight interaction between Hu-GPIbα and rLep-vWA-I/rLep-vWA-II with KD values of 3.87 × 10-7-8.65 × 10-8 M. Hu-GPIbα-binding of rL-vWA-I/rL-vWA-II neither activated the PI3K/AKT-ERK and PLC/PKC kinases nor affected the NO, cGMP, ADP, Ca2+ and TXA2 levels in Hu-platelets. G13/R36/G47 in Lep-vWA-I and G76/Q126 in Lep-vWA-II were confirmed as the Hu-GPIbα-binding sites. Injection of rLep-vWA-I or rLep-vWA-II in mice resulted in diffuse pulmonary and focal renal hemorrhage but this hemorrhage was blocked by rLep-vWA-I-IgG or rLep-vWA-II-IgG. INTERPRETATION: The products of L. interrogans vwa-I and vwa-II genes induce hemorrhage by competitive inhibition of vWF-mediated Hu-platelet aggregation.

A novel Fas-binding outer membrane protein and lipopolysaccharide of Leptospira interrogans induce macrophage apoptosis through the Fas/FasL-caspase-8/-3 pathway.

Leptospira interrogans is the major causative agent of leptospirosis, an emerging, globally spreading zoonotic infectious disease. The pathogen induces macrophage apoptosis, but the molecular basis and mechanism remain unknown. In the present study, we found that L. interrogans caused apoptosis of phagocytosis-inhibited macrophages, and the product of the L. interrogans LB047 gene (Lep-OMP047) was the unique protein captured by mouse and human Fas proteins. The recombinant expressed Lep-OMP047 (rLep-OMP047) strongly bound mouse and human Fas proteins with equilibrium association constant (KD) values of 5.20 × 10-6 to 2.84 × 10-9 M according to surface plasmon resonance measurement and isothermal titration calorimetry. Flow-cytometric examination showed that 5 µg rLep-OMP047 or 1 µg lipopolysaccharide of L. interrogans (Lep-LPS) caused 43.70% or 21.90% early apoptosis in mouse J774A.1 macrophages and 28.41% or 15.80% for PMA-differentiated human THP-1 macrophages, respectively, but the apoptosis was blocked by Fas-antagonizing IgGs, Fas siRNAs, and caspase-8/-3 inhibitors. Moreover, Lep-OMP047 was significantly upregulated during infection of macrophages. Lep-LPS promoted the expression and cytomembrane translocation of Fas and FasL in macrophages. The JNK and p38 MAPK but not ERK signaling pathways, as well as the transcription factors c-Jun and ATF2 but not CHOP, mediated Lep-LPS-induced Fas/FasL expression and translocation. TLR2 but not TLR4 mediated Lep-LPS-induced JNK/p38 MAPK activation. Therefore, we demonstrated that a novel Fas-binding OMP and LPS of L. interrogans induce macrophage apoptosis through the Fas/FasL-caspase-8/-3 pathway.

The sigma factor σ54 is required for the long-term survival of Leptospira biflexa in water.

Leptospira spp. comprise both pathogenic and free-living saprophytic species. Little is known about the environmental adaptation and survival mechanisms of Leptospira. Alternative sigma factor, σ54 (RpoN) is known to play an important role in environmental and host adaptation in many bacteria. In this study, we constructed an rpoN mutant by allele exchange, and the complemented strain in saprophytic L. biflexa. Transcriptome analysis revealed that expression of several genes involved in nitrogen uptake and metabolism, including amtB1, glnB-amtB2, ntrX and narK, were controlled by σ54 . While wild-type L. biflexa could not grow under nitrogen-limiting conditions but was able to survive under such conditions and recover rapidly, the rpoN mutant was not. The rpoN mutant also had dramatically reduced ability to survive long-term in water. σ54 appears to regulate expression of amtB1, glnK-amtB2, ntrX and narK in an indirect manner. However, we identified a novel nitrogen-related gene, LEPBI_I1011, whose expression was directly under the control of σ54 (herein renamed as rcfA for RpoN-controlled factor A). Taken together, our data reveal that the σ54 regulatory network plays an important role in the long-term environmental survival of Leptospira spp.

A leptospiral AAA+ chaperone-Ntn peptidase complex, HslUV, contributes to the intracellular survival of Leptospira interrogans in hosts and the transmission of leptospirosis.

Leptospirosis caused by Leptospira is a zoonotic disease of global importance but it is considered as an emerging or re-emerging infectious disease in many areas in the world. Until now, the mechanisms about pathogenesis and transmission of Leptospira remains poorly understood. As eukaryotic and prokaryotic proteins can be denatured in adverse environments and chaperone-protease/peptidase complexes degrade these harmful proteins, we speculate that infection may also cause leptospiral protein denaturation, and the HslU and HslV proteins of L. interrogans may compose a complex to degrade denatured proteins that enhances leptospiral survival in hosts. Here we show that leptospiral HslUV is an ATP-dependent chaperone-peptidase complex containing ATPase associated with various cellular activity (AAA+) and N-terminal nucleophile (Ntn) hydrolase superfamily domains, respectively, which hydrolyzed casein and chymotrypsin-like substrates, and this hydrolysis was blocked by threonine protease inhibitors. The infection of J774A.1 macrophages caused the increase of leptospiral denatured protein aggresomes, but more aggresomes accumulated in hslUV gene-deleted mutant. The abundant denatured leptospiral proteins are involved in ribosomal structure, flagellar assembly, two-component signaling systems and transmembrane transport. Compared to the wild-type strain, infection of cells in vitro with the mutant resulted in a higher number of dead leptospires, less leptospiral colony-forming units and lower growth ability, but also displayed a lower half lethal dose, attenuated histopathological injury and decreased leptospiral loading in lungs, liver, kidneys, peripheral blood and urine in hamsters. Therefore, our findings confirmed that HslUV AAA+ chaperone-Ntn peptidase complex of L. interrogans contributes to leptospiral survival in hosts and transmission of leptospirosis.

Bid-Induced Release of AIF/EndoG from Mitochondria Causes Apoptosis of Macrophages during Infection with Leptospira interrogans.

Leptospirosis is a global zoonotic infectious disease caused by pathogenic Leptospira species. Leptospire-induced macrophage apoptosis through the Fas/FasL-caspase-8/3 pathway plays an important role in the survival and proliferation of the pathogen in hosts. Although, the release of mitochondrial apoptosis-inducing factor (AIF) and endonuclease G (EndoG) in leptospire-infected macrophages has been described, the mechanisms linking caspase and mitochondrion-related host-cell apoptosis has not been determined. Here, we demonstrated that leptospire-infection induced apoptosis through mitochondrial damages in macrophages. Apoptosis was caused by the mitochondrial release and nuclear translocation of AIF and/or EndoG, leading to nuclear DNA fragmentation. However, the mitochondrion-related CytC-caspase-9/3 pathway was not activated. Next, we found that the release and translocation of AIF and/or EndoG was preceded by the activation of the BH3-interacting domain death agonist (Bid). Furthermore, our data demonstrated that caspase-8 was activated during the infection and caused the activation of Bid. Meanwhile, high reactive oxygen species (ROS) trigged by the infection caused the dephosphorylation of Akt, which also activated Bid. In conclusion, Bid-mediated mitochondrial release of AIF and/or EndoG followed by nuclear translocation is a major mechanism of leptospire- induced apoptosis in macrophages, and this process is modulated by both caspase-8 and ROS-Akt signal pathways.

Mononuclear-macrophages but not neutrophils act as major infiltrating anti-leptospiral phagocytes during leptospirosis.

OBJECTIVE: To identify the major infiltrating phagocytes during leptospirosis and examine the killing mechanism used by the host to eliminate Leptospira interrogans. METHODS: Major infiltrating phagocytes in Leptospira-infected C3H/HeJ mice were detected by immunohistochemistry. Chemokines and vascular endothelial cell adhesion molecules (VECAMs) of Leptospira-infected mice and leptospirosis patients were detected by microarray and immunohistochemistry. Leptospira-phagocytosing and -killing abilities of human or mouse macrophages and neutrophils, and the roles of intracellular ROS, NO and [Ca2+]i in Leptospira-killing process were evaluated by confocal microscopy and spectrofluorimetry. RESULTS: Peripheral blood mononuclear-macrophages rather than neutrophils were the main infiltrating phagocytes in the lungs, liver and kidneys of infected mice. Levels of macrophage- but not neutrophil-specific chemokines and VECAMs were significantly increased in the samples from infected mice and patients. All macrophages tested had a higher ability than neutrophils to phagocytose and kill leptospires. Higher ROS and NO levels and [Ca2+]i in the macrophages were involved in killing leptospires. Human macrophages displayed more phagolysosome formation and a stronger leptospire-killing ability to than mouse macrophages. CONCLUSIONS: Mononuclear-macrophages but not neutrophils represent the main infiltrating and anti-leptospiral phagocytes during leptospirosis. A lower level of phagosome-lysosome fusion may be responsible for the lower Leptospira-killing ability of human macrophages.

Identification of prognostic signature in cancer based on DNA methylation interaction network.

BACKGROUND: The identification of prognostic biomarkers for cancer patients is essential for cancer research. These days, DNA methylation has been proved to be associated with cancer prognosis. However, there are few methods which identify the prognostic markers based on DNA methylation data systematically, especially considering the interaction among DNA methylation sites. METHODS: In this paper, we first evaluated the stabilities of microRNA, mRNA, and DNA methylation data in prognosis of cancer. After that, a rank-based method was applied to construct a DNA methylation interaction network. In this network, nodes with the largest degrees (10% of all the nodes) were selected as hubs. Cox regression was applied to select the hubs as prognostic signature. In this prognostic signature, DNA methylation levels of each DNA methylation site are correlated with the outcomes of cancer patients. After obtaining these prognostic genes, we performed the survival analysis in the training group and the test group to verify the reliability of these genes. RESULTS: We applied our method in three cancers (ovarian cancer, breast cancer and Glioblastoma Multiforme). In all the three cancers, there are more common ones of prognostic genes selected from different samples in DNA methylation data, compared with gene expression data and miRNA expression data, which indicates the DNA methylation data may be more stable in cancer prognosis. Power-law distribution fitting suggests that the DNA methylation interaction networks are scale-free. And the hubs selected from the three networks are all enriched by cancer related pathways. The gene signatures were obtained for the three cancers respectively, and survival analysis shows they can distinguish the outcomes of tumor patients in both the training data sets and test data sets, which outperformed the control signatures. CONCLUSIONS: A computational method was proposed to construct DNA methylation interaction network and this network could be used to select prognostic signatures in cancer.

The EbpA-RpoN Regulatory Pathway of the Pathogen Leptospira interrogans Is Essential for Survival in the Environment.

Leptospira interrogans is the agent of leptospirosis, a reemerging zoonotic disease. It is transmitted to humans through environmental surface waters contaminated by the urine of mammals chronically infected by pathogenic strains able to survive in water for long periods. Little is known about the regulatory pathways underlying environmental sensing and host adaptation of L. interrogans during its enzootic cycle. This study identifies the EbpA-RpoN regulatory pathway in L. interrogans In this pathway, EbpA, a σ54 activator and putative prokaryotic enhancer-binding protein (EBP), and the alternative sigma factor RpoN (σ54) control expression of at least three genes, encoding AmtB (an ammonium transport protein) and two proteins of unknown function. Electrophoresis mobility shift assay demonstrated that recombinant RpoN and EbpA bind to the promoter region and upstream of these three identified genes, respectively. Genetic disruption of ebpA in L. interrogans serovar Manilae virtually abolished expression of the three genes, including amtB in two independent ebpA mutants. Complementation of the ebpA mutant restored expression of these genes. Intraperitoneal inoculation of gerbils with the ebpA mutant did not affect mortality. However, the ebpA mutant had decreased cell length in vitro and had a significantly lowered cell density at stationary phase when grown with l-alanine as the sole nitrogen source. Furthermore, the ebpA mutant has dramatically reduced long-term survival ability in water. Together, these studies identify a regulatory pathway, the EbpA-RpoN pathway, that plays an important role in the zoonotic cycle of L. interrogans IMPORTANCE: Leptospirosis is a reemerging disease with global importance. However, our understanding of gene regulation of the spirochetal pathogen Leptospira interrogans is still in its infancy, largely due to the lack of robust tools for genetic manipulation of this spirochete. Little is known about how the pathogen achieves its long-term survival in the aquatic environment. By utilizing bioinformatic, genetic, and biochemical methods, we discovered a regulatory pathway in L. interrogans, the EbpA-RpoN pathway, and demonstrated that this pathway plays an important role in environmental survival of this pathogen.

Serum Concentrations of Antibodies against Outer Membrane Protein P6, Protein D, and T- and B-Cell Combined Antigenic Epitopes of Nontypeable Haemophilus influenzae in Children and Adults of Different Ages.

Nontypeable Haemophilus influenzae (NTHi) is one of the most common etiologies of acute otitis media, rhinosinusitis, and pneumonia. Outer membrane proteins (OMPs) are the main focus in new vaccine development against NTHi, as the H. influenzae type b (Hib) vaccine does not cover noncapsulated NTHi. The OMPs P6 and protein D are the most promising candidate antigens for an NTHi vaccine, and low antibody levels against them in serum may be correlated with infection caused by NTHi. In the current study, we measured the antibody titers against P6, protein D, and their T- and B-cell combined peptide epitopes in healthy individuals of different ages. We found that children <1 month old had the lowest antibody levels against NTHi P6, protein D, and their T- and B-cell combined antigenic epitopes. Antibody titers increased at ages 1 to 6 months, peaked at 7 months to 3 years, and remained high at 4 to 6 years. The antibody titers started to decrease after 6 years and were the lowest in the 21- to 30-year group. The geometric mean titers (GMTs) of T- and B-cell combined antigenic epitopes in P6 and protein D were positively correlated with those of the protein antigens. Among 12 peptides tested, P6-61, P6-123, and protein D-167 epitopes were better recognized than others in human serum. These findings might contribute to the development of an effective serotype-independent vaccine for H. influenzae.

Eosinophils from Murine Lamina Propria Induce Differentiation of Naïve T Cells into Regulatory T Cells via TGF-ß1 and Retinoic Acid.

Treg cells play a crucial role in immune tolerance, but mechanisms that induce Treg cells are poorly understood. We here have described eosinophils in lamina propria (LP) that displayed high aldehyde dehydrogenase (ALDH) activity, a rate-limiting step during all-trans retinoic acid (ATRA) synthesis, and expressed TGF-ß1 mRNA and high levels of ATRA. Co-incubation assay confirmed that LP eosinophils induced the differentiation of naïve T cells into Treg cells. Differentiation promoted by LP eosinophils were inhibited by blocked either TGF-ß1 or ATRA. Peripheral blood (PB) eosinophils did not produce ATRA and could not induce Treg differentiation. These data identifies LP eosinophils as effective inducers of Treg cell differentiation through a mechanism dependent on TGF-ß1 and ATRA.

Effects of oxidized low density lipoprotein on transformation of valvular myofibroblasts to osteoblast-like phenotype.

In order to investigate the roles of Wnt signal pathway in transformation of cardiac valvular myofibroblasts to the osteoblast-like phenotype, the primary cultured porcine aortic valve myofibroblasts were incubated with oxidized low density lipoprotein (ox-LDL, 50 mg/L), and divided into four groups according to the ox-LDL treatment time: control group, ox-LDL 24-h group, ox-LDL 48-h group, and ox-LDL 72-h group. Wnt signal pathway blocker Dickkopf-1 (DDK-1, 100 µg/L) was added in ox-LDL 72-h group. The expression of a-smooth muscle actin (α-SMA), bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP), and osteogenic transcription factor Cbfa-1 was detected by Western blotting, and that of ß-catenin, a key mediator of Wnt signal pathway by immunocytochemical staining method. The Wnt/ß-catenin was observed and the transformation of myofibroblasts to the osteoblast-like phenotype was examined. The expression of α-SMA, BMP2, ALP and Cbfa-1 proteins in the control group was weaker than in the ox-LDL-treated groups. In ox-LDL-treated groups, the protein expression of a-SMA, BMP2, ALP, and Cbfa-1 was significantly increased in a time-dependent manner as compared with the control group, and there was significant difference among the three ox-LDL-treated groups (P<0.05 for all); ß-catenin protein was also up-regulated in the ox-LDL-treated groups in a time-dependent manner as compared with the control group (P<0.05), and its transfer from cytoplasm to nucleus and accumulation in the nucleus were increased in the same fashion (P<0.05). After addition of DKK-1, the expression of α-SMA, bone-related proteins and ß-catenin protein was significantly reduced as compared with ox-LDL 72-h group (P<0.05). The Wnt/ ß-catenin signaling pathway may play an important role in transformation of valvular myofibroblasts to the osteoblast-like phenotype.

[Mechanism of tanshinone II A in inhibiting transformation of aortic valvular myofibroblast to osteoblast-like phenotype].

Aortic valve calcification (AVC) is a pathological process correlated with multiple disease causes and actively regulated by cardiac valve cells. In this study, porcine aortic valve myofibroblasts cultured in vitro were treated with 50 µg z L(-1) of pathological factor tumor necrosis factor α (TNF-α). Tanshinone II A (TSN) with the concentration of 50 mg x L(-1) and TNF-α were combined in incubating cells for 72 h (3 d) and 120 h (5 d). The Western blotting and Real-time PCR were adopted to detect the changes in smooth muscle α actin (α-SMA), bone morphogenetic protein 2 ( BMP2), alkaline phosphatase (ALP) in cells, and expressions of key effect proteins GSK-3ß and ß-catenin on Wnt/ß-catenin signal pathway. According to the findings, TNF-α can significantly increase the expression of myofibroblasts α-SMA and add the transformation activity to them, with nearly no expression of BMP2, ALP and mRNA in the control group and the TSN group but significant increase in their expressions in the TNF-α group (P < 0.01), which showed osteoblast-like phenotype. Moreover, TNF-α down-regulated the expression of up-streaming regulator GSK-3ß and mRNA expression (P < 0. 01) , notably increased the expression of key effect protein ß-catenin, but with no significant difference in mRNA with the control group and the TSN group. The result demonstrated that TSN showed a certain inhibitory effect on TNF-α's pathological impact (P < 0.05) in a time-dependent manner. Inflammatory factor TNF-α may promote the transformation of aortic valvular myofibroblasts to osteoblast-like phenotype by activating Wnt/ß-catenin signal pathway in aortic valvular myofibroblasts, so as to cause AVC. Tanshinone II A can have a preventive effect in AVC by activating GSK-3ß proteins and regulating signal transduction of Wnt/ß-catenin signal pathway.