Human induced pluripotent stem cells are resistant to human cytomegalovirus infection primarily at the attachment level due to the reduced expression of cell-surface heparan sulfate.

Cytomegalovirus (CMV), a type of herpes virus, is the predominant cause of congenital anomalies due to intrauterine infections in humans. Adverse outcomes related to intrauterine infections with human cytomegalovirus (HCMV) vary widely, depending on factors such as fetal infection timing, infection route, and viral virulence. The precise mechanism underlying HCMV susceptibility remains unclear. In this study, we compared the susceptibility of neonatal human dermal fibroblast cells (NHDFCs) and human induced pluripotent stem cells (hiPSCs) derived from NHDFCs, which are genetically identical to HCMV, using immunostaining, microarray, in situ hybridization, quantitative PCR, and scanning electron microscopy. These cells were previously used to compare CMV susceptibility, but the underlying mechanisms were not fully elucidated. HCMV susceptibility of hiPSCs was significantly lower in the earliest phase. No shared gene ontologies were observed immediately post-infection between the two cell types using microarray analysis. Early-stage expression of HCMV antigens and the HCMV genome was minimal in immunostaining and in in situ hybridization in hiPSCs. This strongly suggests that HCMV does not readily bind to hiPSC surfaces. Scanning electron microscopy performed using the NanoSuit method confirmed the scarcity of HCMV particles on hiPSC surfaces. The zeta potential and charge mapping of the charged surface in NHDFCs and hiPSCs exhibited minimal differences when assessed using zeta potential analyzer and scanning ion conductance microscopy; however, the expression of heparan sulfate (HS) was significantly lower in hiPSCs compared with that in NHDFCs. Thus, HS expression could be a primary determinant of HCMV resistance in hiPSCs at the attachment level. IMPORTANCE: Numerous factors such as attachment, virus particle entry, transcription, and virus particle egress can affect viral susceptibility. Since 1984, pluripotent cells are known to be CMV resistant; however, the exact mechanism underlying this resistance remains elusive. Some researchers suggest inhibition in the initial phase of HCMV binding, while others have suggested the possibility of a sufficient amount of HCMV entering the cells to establish latency. This study demonstrates that HCMV particles rarely attach to the surfaces of hiPSCs. This is not due to limitations in the electrostatic interactions between the surface of hiPSCs and HCMV particles, but due to HS expression. Therefore, HS expression should be recognized as a key factor in determining the susceptibility of HCMV in congenital infection in vitro and in vivo. In the future, drugs targeting HS may become crucial for the treatment of congenital CMV infections. Thus, further research in this area is warranted.

Role of hepcidin upregulation and proteolytic cleavage of ferroportin 1 in hepatitis C virus-induced iron accumulation.

Hepatitis C virus (HCV) is a pathogen characterized not only by its persistent infection leading to the development of cirrhosis and hepatocellular carcinoma (HCC), but also by metabolic disorders such as lipid and iron dysregulation. Elevated iron load is commonly observed in the livers of patients with chronic hepatitis C, and hepatic iron overload is a highly profibrogenic and carcinogenic factor that increases the risk of HCC. However, the underlying mechanisms of elevated iron accumulation in HCV-infected livers remain to be fully elucidated. Here, we observed iron accumulation in cells and liver tissues under HCV infection and in mice expressing viral proteins from recombinant adenoviruses. We established two molecular mechanisms that contribute to increased iron load in cells caused by HCV infection. One is the transcriptional induction of hepcidin, the key hormone for modulating iron homeostasis. The transcription factor cAMP-responsive element-binding protein hepatocyte specific (CREBH), which was activated by HCV infection, not only directly recognizes the hepcidin promoter but also induces bone morphogenetic protein 6 (BMP6) expression, resulting in an activated BMP-SMAD pathway that enhances hepcidin promoter activity. The other is post-translational regulation of the iron-exporting membrane protein ferroportin 1 (FPN1), which is cleaved between residues Cys284 and Ala285 in the intracytoplasmic loop region of the central portion mediated by HCV NS3-4A serine protease. We propose that host transcriptional activation triggered by endoplasmic reticulum stress and FPN1 cleavage by viral protease work in concert to impair iron efflux, leading to iron accumulation in HCV-infected cells.

Identifying Active Progeny Virus Particles in Formalin-Fixed, Paraffin-Embedded Sections Using Correlative Light and Scanning Electron Microscopy.

Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) tissue blocks is routinely used to identify virus-infected cells. However, detecting virus particles in FFPE sections using light microscopy is difficult because of the light diffraction resolution limitations of an optical microscope. In this study, light microscopy and field emission scanning electron microscopy were performed to observe 3-dimensional virus particles in FFPE sections in a nondestructive manner using NanoSuit or osmium conductive treatment methods. The virus particles in FFPE sections were immunostained with specific antibodies against the surface antigens of the viral particles and stained with 3,3'-diaminobenzidine. A metal solution (0.2% gold chloride or 2% osmium tetroxide) was applied to enhance the 3,3'-diaminobenzidine-stained area. This procedure is nondestructive for FFPE sections and is a simpler method than transmission electron microscopy. To validate the applicability of this technique, we performed 3-dimensional imaging of the virus particles of different sizes, such as human papillomavirus, cytomegalovirus, and varicella-zoster virus. Furthermore, ultrathin sections from the FFPE sections that were observed to harbor viral particles using field emission scanning electron microscopy were prepared and assessed using transmission electron microscopy. In the correlative areas, transmission electron microscopy confirmed the presence of large numbers of virus particles. These results indicated that the combination of marking viral particles with 3,3'-diaminobenzidine/metal staining and conductive treatment can identify active progeny virus particles in FFPE sections using scanning electron microscopy. This easy correlative imaging of field emission scanning electron microscopy of the identical area of FFPE in light microscopy may help elucidate new pathological mechanisms of virus-related diseases.

Primary Cilia Are Frequently Present in Small Cell Lung Carcinomas but Not in Non-Small Cell Lung Carcinomas or Lung Carcinoids.

Most human malignant neoplasms show loss of primary cilia (PC). However, PC are known to be retained and involved in tumorigenesis in some types of neoplasms. The PC status in lung carcinomas remains largely uninvestigated. In this study, we comprehensively assessed the PC status in lung carcinomas. A total of 492 lung carcinomas, consisting of adenocarcinomas (ACs) (n = 319), squamous cell carcinomas (SCCs) (n = 152), and small cell lung carcinomas (SCLCs) (n = 21), were examined by immunohistochemical analysis using an antibody against ARL13B, a marker of PC. The PC-positive rate was markedly higher in SCLCs (81.0%) than in ACs (1.6%) and SCCs (7.9%). We subsequently performed analyses to characterize the PC-positive lung carcinomas further. PC-positive lung carcinomas were more numerous and had longer PC than normal cells. The presence of PC in these cells was not associated with the phase of the cell cycle. We also found that the PC were retained even in metastases from PC-positive lung carcinomas. Furthermore, the hedgehog signaling pathway was activated in PC-positive lung carcinomas. Because ARL13B immunohistochemistry of lung carcinoids (n = 10) also showed a statistically significantly lower rate (10.0%) of PC positivity than SCLCs, we searched for a gene(s) that might be upregulated in PC-positive SCLCs compared with lung carcinoids, but not in PC-negative carcinomas. This search, and further cell culture experiments, identified HYLS1 as a gene possessing the ability to regulate ciliogenesis in PC-positive lung carcinomas. In conclusion, our findings indicate that PC are frequently present in SCLCs but not in non-SCLCs (ACs and SCCs) or lung carcinoids, and their PC exhibit various specific pathobiological characteristics. This suggests an important link between lung carcinogenesis and PC.

Carbon nanodots with a controlled N structure by a solvothermal method for generation of reactive oxygen species under visible light.

Carbon nanodots can function as photosensitizers that have the ability to generate reactive oxygen species such as singlet oxygen, hydroxy (OH) radicals, and superoxide ions. However, most of these can only be generated upon ultraviolet light excitation. Additionally, the mechanism of reactive oxygen species generation by carbon nanodots remains unclear. The development of carbon nanodots that can photosensitize under visible light irradiation is desirable for applications such as photodynamic therapy and pollutant decomposition under visible light. Here, we report novel carbon nanodot-based photosensitizers that generate reactive oxygen species under visible light; they were synthesized using a solvothermal method with two solvents (formamide and water) and amidol as the carbon source. Carbon nanodots from the solvothermal synthesis in formamide showed blue fluorescence, while those obtained in water showed green fluorescence. The photo-excited blue-fluorescent carbon nanodots produced OH radicals, superoxide ions, and singlet oxygen, and therefore could function as both type I and type II photosensitizers. In addition, photo-excited green-fluorescent carbon nanodots generated only singlet oxygen, therefore functioning as type II photosensitizers. It is proposed that the two photosensitizers have different origins of reactive oxygen species generation: the enrichment of graphitic N for blue-fluorescent carbon nanodots and molecular fluorophores for green-fluorescent carbon nanodots.

Identification and characterization of primary cilia-positive salivary gland tumours exhibiting basaloid/myoepithelial differentiation.

Primary cilia (PC) are non-motile, antenna-like structures on the cell surface. Many types of neoplasms exhibit PC loss, whereas in some neoplasms PC are retained and involved in tumourigenesis. To elucidate the PC status and characteristics of major salivary gland tumours (SGTs), we examined 100 major SGTs encompassing eight histopathological types by immunohistochemical analysis. PC were present in all (100%) of the pleomorphic adenomas (PAs), basal cell adenomas (BCAs), adenoid cystic carcinomas (AdCCs), and basal cell adenocarcinomas (BCAcs) examined, but absent in all (0%) of the Warthin tumours, salivary duct carcinomas, mucoepidermoid carcinomas, and acinic cell carcinomas examined. PC were also detected by electron-microscopic analysis using the NanoSuit method. It is worthy of note that the former category and latter category of tumours contained and did not contain a basaloid/myoepithelial differentiation component, respectively. The four types of PC-positive SGTs showed longer PC than normal and exhibited a characteristic distribution pattern of the PC in the ductal and basaloid/neoplastic myoepithelial components. Two PC-positive carcinomas (AdCC and BCAc) still possessed PC in their recurrent/metastatic sites. Interestingly, activation of the Hedgehog signalling pathway, shown by predominantly nuclear GLI1 expression, was significantly more frequently observed in PC-positive SGTs. Finally, we identified tau tubulin kinase 2 (TTBK2) as being possibly involved in the production of PC in SGTs. Taken together, our findings indicate that SGTs that exhibit basaloid/myoepithelial differentiation (PA, BCA, AdCC, and BCAc) are ciliated, and their PC exhibit tumour-specific characteristics, are involved in activation of the Hedgehog pathway, and are associated with TTBK2 upregulation, providing a significant and important link between SGT tumourigenesis and PC. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Exosomes from dental pulp cells attenuate bone loss in mouse experimental periodontitis.

BACKGROUND AND OBJECTIVE: Exosomes are small vesicles secreted from many cell types. Their biological effects largely depend on their cellular origin and the physiological state of the originating cells. Exosomes secreted by mesenchymal stem cells exert therapeutic effects against multiple diseases and may serve as potential alternatives to stem cell therapies. We previously established and characterized human leukocyte antigen (HLA) haplotype homo (HHH) dental pulp cell (DPC) lines from human wisdom teeth. In this study, we aimed to investigate the effect of local administration of HHH-DPC exosomes in a mouse model of periodontitis. METHODS: Exosomes purified from HHH-DPCs were subjected to particle size analysis, and expression of exosome markers was confirmed by western blotting. We also confirmed the effect of exosomes on the migration of both HHH-DPCs and mouse osteoblastic MC3T3-E1 cells. A mouse experimental periodontitis model was used to evaluate the effect of exosomes in vivo. The morphology of alveolar bone was assessed by micro-computed tomography (µCT) and histological analysis. The effect of exosomes on osteoclastogenesis was evaluated using a co-culture system. RESULTS: The exosomes purified from HHH-DPCs were homogeneous and had a spherical membrane structure. HHH-DPC exosomes promoted the migration of both human DPCs and mouse osteoblastic cells. The MTT assay showed a positive effect on the proliferation of human DPCs, but not on mouse osteoblastic cells. Treatment with HHH-DPC exosomes did not alter the differentiation of osteoblastic cells. Imaging with µCT revealed that the exosomes suppressed alveolar bone resorption in the mouse model of periodontitis. Although no change was apparent in the dominance of TRAP-positive osteoclast-like cells in decalcified tissue sections upon exosome treatment, HHH-DPC exosomes significantly suppressed osteoclast formation in vitro. CONCLUSIONS: HHH-DPC exosomes stimulated the migration of human DPCs and mouse osteoblastic cells and effectively attenuated bone loss due to periodontitis.

Difference in the distribution of tumor-infiltrating CD8+ T cells and FOXP3+ T cells between micronodular thymoma with lymphoid stroma and micronodular thymic carcinoma with lymphoid stroma.

Micronodular thymoma with lymphoid stroma (MNT) is a rare thymic epithelial neoplasm subtype characterized by a micronodular tumor cell growth pattern and abundant lymphoid stroma. Micronodular thymic carcinoma with lymphoid stroma (MNCA) is considered as a malignant counterpart of MNT and exhibits a growth pattern similar to that of MNT but has histologic features reminiscent of thymic squamous cell carcinoma, such as cytologic atypia and CD5 and CD117 immunoexpression. Although both MNT and MNCA are characterized by abundant lymphoid stroma, it remains unknown whether there are differences in infiltrating lymphocytes between MNT and MNCA. We analyzed the immune microenvironment profile in eight MNT and three MNCA cases. The cell density of CD8-positive T cells was significantly higher in MNT than in MNCA, whereas that of FOXP3-positive T cells was significantly higher in MNCA than in MNT. There was no significant difference in the cell density of programmed death protein 1-positive T cells and programmed death ligand 1 expression between the MNT and MNCA cases. Our findings indicated that the immune microenvironment of MNCA differed from that of MNT and, compared with the T-cell profile of MNT, that of MNCA was more suppressive to patients' antitumor immune response.

TiO2-supported Au144 nanoclusters for enhanced sonocatalytic performance.

The production of reactive oxygen species (ROS), such as hydroxyl radicals, by ultrasonic activation of semiconductor nanoparticles (NPs), including TiO2, has excellent potential for use in sonodynamic therapy and for the sonocatalytic degradation of pollutants. However, TiO2 NPs have limitations including low yields of generated ROS that result from fast electron-hole recombination. In this study, we first investigated the sonocatalytic activity of TiO2-supported Au nanoclusters (NCs) (Au NCs/TiO2) by monitoring the production of hydroxyl radicals (•OH) under ultrasonication conditions. The deposition of Au144 NCs on TiO2 NPs was found to enhance sonocatalytic activity for •OH production by approximately a factor of 2. Electron-hole recombination in ultrasonically excited TiO2 NPs is suppressed by Au144 NCs acting as an electron trap; this charge separation resulted in enhanced •OH production. In contrast, the deposition of Au25 NCs on TiO2 NPs resulted in lower sonocatalytic activity due to less charge separation, which highlights the effectiveness of combining Au144 NCs with TiO2 NPs for enhancing sonocatalytic activity. The sonocatalytic action that forms electron-hole pairs on the Au144/TiO2 catalyst is due to both heat and sonoluminescence from the implosive collapse of cavitation bubbles. Consequently, the ultrasonically excited Au144 (3 wt. %)/TiO2 catalyst exhibited higher catalytic activity for the production of •OH because of less light shadowing effect, in contrast to the lower catalytic activity when irradiated with only external light.

Gremlin-1 for the Differential Diagnosis of Idiopathic Pulmonary Fibrosis Versus Other Interstitial Lung Diseases: A Clinical and Pathophysiological Analysis.

PURPOSE: The differential diagnosis of interstitial lung diseases (ILDs), particularly idiopathic pulmonary fibrosis (IPF) versus other non-IPF ILDs, is important for selecting the appropriate treatment. This retrospective study aimed to explore the utility of gremlin-1 for the differential diagnosis. METHODS: Serum gremlin-1 concentrations were measured using an ELISA in 50 patients with IPF, 42 patients with non-IPF ILD, and 30 healthy controls. The baseline clinical data, including pulmonary functions, prognosis, and three serum biomarkers (Krebs von den Lungen-6 [KL6], surfactant protein-D [SP-D], and lactate dehydrogenase [LDH]), were obtained through a medical record review for analyzing their associations with serum gremlin-1 concentrations. To evaluate the origin of gremlin-1, we performed immunostaining on lung sections. RESULTS: Serum gremlin-1 concentrations were significantly higher in patients with IPF (mean concentration, 14.4 ng/mL), followed by those with non-IPF ILD (8.8 ng/mL) and healthy controls (1.6 ng/mL). The area under the curve for IPF versus non-IPF ILDs was 0.759 (95% confidence interval, 0.661-0.857), which was superior to that of KL6/SP-D/LDH. The sensitivity and specificity for gremlin-1 (cutoff, 10.4 ng/mL) was 72 and 69%, respectively. By contrast, serum gremlin-1 concentrations were not associated with the pulmonary functions nor the prognosis in all patients with ILDs. In immunostaining, the gremlin-1 was broadly upregulated in IPF lungs, particularly at myofibroblasts, bronchiolar/alveolar epithelium, and CD163-positive M2-like macrophages. CONCLUSIONS: Gremlin-1 may be a useful biomarker to improve the diagnostic accuracy for IPF compared to non-IPF ILDs, suggesting a role of this molecule in the pathogenesis of IPF.

The NanoSuit method: a novel histological approach for examining paraffin sections in a nondestructive manner by correlative light and electron microscopy.

Histological examination using the light microscopy is currently the gold standard for life science research and diagnostics. However, magnified observations are limited because of the limitations intrinsic to light microscopy. Thus, a dual approach, known as correlative light and electron microscopy (CLEM), has emerged, although several technical challenges remain in terms of observing myriad stored paraffin sections. Previously, we developed the NanoSuit method, which enabled us to keep multicellular organisms alive/wet in the high vacuum of a scanning electron microscope by encasing the sample in a thin, vacuum-proof membrane. The approach uses the native extracellular substance (ECS) or an ECS-mimicking substance to polymerize a membrane by plasma or electron beam irradiation. Since the resulting NanoSuit is flexible and dense enough to prevent a living organism's bodily gas and liquids from evaporating (which we refer to as the "surface shield enhancer" (SSE) effect), it works like a miniature spacesuit with sufficient electron conductivity for an SEM observation. Here, we apply the NanoSuit method to CLEM analysis of paraffin sections. Accordingly, the NanoSuit method permits the study of paraffin sections using CLEM at low and high magnification, with the following features: (i) the integrity of the glass slide is maintained, (ii) three-dimensional microstructures of tissue and pathogens are visualized, (iii) nuclei and 3,3'-diaminobenzidine-stained areas are distinct because of gold chloride usage, (iv) immunohistochemical staining is quantitative, and (v) contained elements can be analyzed. Removal of the SSE solution after observation is a further advantage, as this allows slides to be restained and stored. Thus, the NanoSuit method represents a novel approach that will advance the field of histology.

CD248 and integrin alpha-8 are candidate markers for differentiating lung fibroblast subtypes.

BACKGROUND: Lung fibrosis is a serious life-threatening condition whose manifestation varies according to the localization and characteristics of fibroblasts, which are considered heterogeneous. Therefore, to better understand the pathology and improve diagnosis and treatment of this disease, it is necessary to elucidate the nature of this heterogeneity and identify markers for the accurate classification of human lung fibroblast subtypes. METHODS: We characterized distinct mouse lung fibroblast subpopulations isolated by fluorescence-activated cell sorting (FACS) and performed microarray analysis to identify molecular markers that could be useful for human lung fibroblast classification. Based on the expression of these markers, we evaluated the fibroblast-like cell subtype localization in normal human lung samples and lung samples from patients with idiopathic pulmonary fibrosis (IPF). RESULTS: Mouse lung fibroblasts were classified into Sca-1high fibroblasts and Sca-1low fibroblasts by in vitro biological analyses. Through microarray analysis, we demonstrated CD248 and integrin alpha-8 (ITGA8) as cell surface markers for Sca-1high fibroblasts and Sca-1low fibroblasts, respectively. In mouse lungs, Sca-1high fibroblasts and Sca-1low fibroblasts were localized in the collagen fiber-rich connective tissue and elastic fiber-rich connective tissue, respectively. In normal human lungs and IPF lungs, two corresponding major fibroblast-like cell subtypes were identified: CD248highITGA8low fibroblast-like cells and CD248lowITGA8high fibroblast-like cells, localized in the collagen fiber-rich connective tissue and in the elastic fiber-rich connective tissue, respectively. CONCLUSION: CD248highITGA8low fibroblast-like cells and CD248lowITGA8high fibroblast-like cells were localized in an almost exclusive manner in human lung specimens. This human lung fibroblast classification using two cell surface markers may be helpful for further detailed investigations of the functions of lung fibroblast subtypes, which can provide new insights into lung development and the pathological processes underlying fibrotic lung diseases.

Label-Free Specific Detection and Collection of C-Reactive Protein Using Zwitterionic Phosphorylcholine-Polymer-Protected Magnetic Nanoparticles.

In this study, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)]-protected Fe3O4 nanoparticles were prepared and used for the label-free specific detection and collection of an acute inflammation marker, C-reactive protein (CRP), in a simulated body fluid. The Fe3O4 nanoparticle surface was modified using poly(MPC) by surface-initiated atom-transfer radical polymerization. The density of poly(MPC) was 0.16 chains/nm2, and the colloidal stability of the nanoparticles in aqueous media and human plasma was effectively improved by surface modification. The size of the as-prepared poly(MPC)-protected Fe3O4 nanoparticles was ∼200 nm. After coming into contact with CRP, the nanoparticles aggregated as CRP comprises five subunits, and each subunit can bind to a phosphorylcholine group with two free Ca2+ ions. The change in the nanoparticle size exhibited a good correlation with the CRP concentration in the range of 0-600 nM. A low limit of detection of 10 nM for CRP was observed. The particles effectively reduced the adsorption of nonspecific proteins, and the change in the nanoparticle size with CRP was not affected by the coexistence of bovine serum albumin at a concentration 1000 times greater than that of CRP. Nanoparticle aggregates formed using CRP were dissociated using ethylenediamine- N, N, N', N'-tetraacetic acid, disodium salt, thereby regenerating poly(MPC)-protected Fe3O4 nanoparticles. In addition, CRP was collected from aqueous media using an acidic buffer solution and human plasma. CRP-containing aqueous solutions were treated with poly(MPC)-protected Fe3O4. After poly(MPC)-protected Fe3O4 nanoparticles were separated using a neodymium magnet and centrifugation, the concentration of CRP in the media dramatically decreased. In stark contrast, the concentration of albumin present in the test solution did not change even after treatment with the nanoparticles. Therefore, nanoparticles specifically recognize CRP from complex biological fluids. Although inhibition tests in the presence of 1,2-dioleoyl- sn-glycero-3-phosphocholine liposomes or free poly(MPC) were also carried out, the binding of poly(MPC)-protected Fe3O4 to CRP was not affected by these inhibitors. In conclusion, poly(MPC)-brush-bearing magnetic nanoparticles can serve not only as reliable materials for detecting and controlling the levels of CRP in simulated body fluids but also as diagnostic and therapeutic materials.

A nanocomposite of N-doped carbon dots with gold nanoparticles for visible light active photosensitisers.

This study aims to report the synthesis of nitrogen-doped carbon dots (N-CDs) derived from biomass, tannic acid and polyethyleneimine, which is a N-doping agent. The N-doping of CDs is found to enhance singlet oxygen (1O2) generation under visible light irradiation. Additionally, the N-CDs work as both a reducing agent and stabiliser, and hence the N-CDs can directly reduce chloroauric acid to form gold nanoparticles (Au NPs) without adding other reducers and stabilisers. The as-prepared N-CD-Au NP nanocomposite possesses 2.3 times higher photosensitising ability for producing 1O2 than N-CDs. To the best of our knowledge, this is the first report on the enhancement of the photosensitising ability of N-CDs by Au NPs. We suggest that the plasmon coupling of Au NPs with the N-CDs causes the energy transfer from the N-CDs to the ground state oxygen (3O2), thus resulting in an increased 1O2 generation.

Mitochondrial fission protein, dynamin-related protein 1, contributes to the promotion of hypertensive cardiac hypertrophy and fibrosis in Dahl-salt sensitive rats.

BACKGROUND: Hypertension promotes cardiac hypertrophy which finally leads to cardiac dysfunction. Although aberrant mitochondrial dynamics is known to be a relevant contributor of pathogenesis in heart disease, little is known about the relationship between mitochondrial dynamics and cardiac hypertrophy. We investigated the pathophysiological roles of Dynamin-related protein1 (Drp1, a mitochondrial fission protein) on the hypertensive cardiac hypertrophy. METHODS & RESULTS: Dahl salt-sensitive rats were fed with a low-salt (0.3% NaCl) or a high-salt (8% NaCl) chow to promote hypertension with and without administration of mdivi1 (an inhibitor of Drp1: 1 mg/kg/every alternative day), and then the hypertensive cardiac hypertrophy was assessed. High-salt fed rats exhibited left ventricular hypertrophy (LVH), myocytes hypertrophy, and cardiac fibrosis, and mdivi-1 suppressed them without alteration of the blood pressure. Mdivi1 also reduced ROS production by hypertension, which subsequently suppressed the Ca2+-activated protein phosphatase calcineurin and Ca2+/calmodulin-dependent kinase II (CaMKII). CONCLUSIONS: Our results suggest that Drp1 contributes to the pathogenesis of hypertensive cardiac hypertrophy via ROS production and the Drp1 suppression may be effective to prevent the hypertensive cardiac hypertrophy.

Podoplanin-positive myofibroblasts: a pathological hallmark of pleuroparenchymal fibroelastosis.

Pathological differential diagnoses of pleuroparenchymal fibroelastosis (PPFE) include usual interstitial pneumonia (UIP) and pulmonary apical cap (PAC); however, there are no specific immunostaining makers to distinguish between these diseases. We performed immunohistochemistry using several pleural mesothelial cell-related markers, including cytokeratin-5/6, CAM5.2, WT-1, calretinin, desmin and podoplanin, for PPFE (n = 4), UIP (n = 10) and PAC (n = 3) lung sections. Among the examined markers, in PPFE and PAC lungs podoplanin commonly showed positivity for spindle cells both in thickened pleura and subpleural fibroelastosis lesions; these cells were also stained with α-smooth muscle actin, a marker of myofibroblasts. However, even in elastic fibre-rich cases, UIP lungs did not show such podoplanin-positive myofibroblasts in pleura/subpleura and fibroblastic foci. These findings were also verified using immunofluorescence. By contrast, immunohistochemically as well as morphologically, the difference between PPFE and PAC was not apparent. The presence of podoplanin-positive myofibroblasts could be a pathological hallmark of PPFE, suggesting a pathogenic process distinct from UIP but common to PAC.

LTBP2 is secreted from lung myofibroblasts and is a potential biomarker for idiopathic pulmonary fibrosis.

Although differentiation of lung fibroblasts into α-smooth muscle actin (αSMA)-positive myofibroblasts is important in the progression of idiopathic pulmonary fibrosis (IPF), few biomarkers reflecting the fibrotic process have been discovered. We performed microarray analyses between FACS-sorted steady-state fibroblasts (lineage (CD45, TER-119, CD324, CD31, LYVE-1, and CD146)-negative and PDGFRα-positive cells) from untreated mouse lungs and myofibroblasts (lineage-negative, Sca-1-negative, and CD49e-positive cells) from bleomycin-treated mouse lungs. Amongst several genes up-regulated in the FACS-sorted myofibroblasts, we focussed on Ltbp2, the gene encoding latent transforming growth factor-ß (TGF-ß) binding protein-2 (LTBP2), because of the signal similarity to Acta2, which encodes αSMA, in the clustering analysis. The up-regulation was reproduced at the mRNA and protein levels in human lung myofibroblasts induced by TGF-ß1. LTBP2 staining in IPF lungs was broadly positive in the fibrotic interstitium, mainly as an extracellular matrix (ECM) protein; however, some of the αSMA-positive myofibroblasts were also stained. Serum LTBP2 concentrations, evaluated using ELISA, in IPF patients were significantly higher than those in healthy volunteers (mean: 21.4 compared with 12.4 ng/ml) and showed a negative correlation with % predicted forced vital capacity (r = -0.369). The Cox hazard model demonstrated that serum LTBP2 could predict the prognosis of IPF patients (hazard ratio for death by respiratory events: 1.040, 95% confidence interval: 1.026-1.054), which was validated using the bootstrap method with 1000-fold replication. LTBP2 is a potential prognostic blood biomarker that may reflect the level of differentiation of lung fibroblasts into myofibroblasts in IPF.

Aberrant Epigenetic Regulation in Head and Neck Cancer Due to Distinct EZH2 Overexpression and DNA Hypermethylation.

Enhancer of Zeste homologue 2 (EZH2) overexpression is associated with tumor proliferation, metastasis, and poor prognosis. Targeting and inhibition of EZH2 is a potentially effective therapeutic strategy for head and neck squamous cell carcinoma (HNSCC). We analyzed EZH2 mRNA expression in a well-characterized dataset of 230 (110 original and 120 validation cohorts) human head and neck cancer samples. This study aimed to investigate the effects of inhibiting EZH2, either via RNA interference or via pharmacotherapy, on HNSCC growth. EZH2 upregulation was significantly correlated with recurrence (p < 0.001) and the methylation index of tumor suppressor genes (p < 0.05). DNMT3A was significantly upregulated upon EZH2 upregulation (p = 0.043). Univariate analysis revealed that EZH2 upregulation was associated with poor disease-free survival (log-rank test, p < 0.001). In multivariate analysis, EZH2 upregulation was evaluated as a significant independent prognostic factor of disease-free survival (hazard ratio: 2.085, 95% confidence interval: 1.390⁻3.127; p < 0.001). Cells treated with RNA interference and DZNep, an EZH2 inhibitor, showed the most dramatic changes in expression, accompanied with a reduction in the growth and survival of FaDu cells. These findings suggest that EZH2 upregulation is correlated with tumor aggressiveness and adverse patient outcomes in HNSCC. Evaluation of EZH2 expression might help predict the prognosis of HNSCC patients.

Evaluation of epigenetic inactivation of vascular endothelial growth factor receptors in head and neck squamous cell carcinoma.

The aim of this study was to determine the methylation status of the genes encoding the vascular endothelial growth factor receptors and to evaluate the usefulness of VEGFR methylation as a prognostic indicator in head and neck squamous cell carcinoma. VEGFR messenger RNA expression and promoter methylation were examined in a panel of cell lines via quantitative reverse transcription and methylation-specific polymerase chain reaction, respectively. Promoter methylation was compared with clinical characteristics in 128 head and neck squamous cell carcinoma samples. The normalized methylation values for the VEGFR1, VEGFR2 and VEGFR3 promoters tended to be higher in the tumour cell lines than in normal tonsil samples, whereas amounts of VEGFR1, VEGFR2 and VEGFR3 messenger RNA were significantly higher. Methylation of the VEGFR1 promoter (p = 0.003; 66/128 head and neck squamous cell carcinoma samples, 52%) and VEGFR3 promoter (p = 0.043; 53/128 head and neck squamous cell carcinoma samples, 41%) significantly correlated with recurrence, whereas methylation of the VEGFR2 promoter significantly correlated with lymph node metastasis (p = 0.046; 47/128 head and neck squamous cell carcinoma samples, 37%). Concurrent methylation of the VEGFR1 and VEGFR3 promoters significantly correlated with reduced disease-free survival (log-rank test, p = 0.009). In a multivariate logistic regression analysis, methylation of the VEGFR1, VEGFR3 and both the VEGFR1 and VEGFR3 promoters independently predicted recurrence (odds ratios and 95% confidence intervals: 3.19, 1.51-6.75 (p = 0.002); 2.24, 1.06-4.76 (p = 0.035); and 2.56, 1.09-6.05 (p = 0.032), respectively). Methylation of the VEGFR promoters predicts poor prognosis in head and neck squamous cell carcinoma patients.

Pathogenesis of developmental anomalies of the central nervous system induced by congenital cytomegalovirus infection.

In humans, the herpes virus family member cytomegalovirus (CMV) is the most prevalent mediator of intrauterine infection-induced congenital defect. Central nervous system (CNS) dysfunction is a distinguishing symptom of CMV infection, and characterized by ventriculoencephalitis and microglial nodular encephalitis. Reports on the initial distribution of CMV particles and its receptors on the blood brain barrier (BBB) are rare. Nevertheless, several factors are suggested to affect CMV etiology. Viral particle size is the primary factor in determining the pattern of CNS infections, followed by the expression of integrin ß1 in endothelial cells, pericytes, meninges, choroid plexus, and neural stem progenitor cells (NSPCs), which are the primary targets of CMV infection. After initial infection, CMV disrupts BBB structural integrity to facilitate the spread of viral particles into parenchyma. Then, the initial meningitis and vasculitis eventually reaches NSPC-dense areas such as ventricular zone and subventricular zone, where viral infection inhibits NSPC proliferation and differentiation and results in neuronal cell loss. These cellular events clinically manifest as brain malformations such as a microcephaly. The purpose of this review is to clearly delineate the pathophysiological basis of congenital CNS anomalies caused by CMV.