The HEDGEHOG-GLI1 pathway is important for fibroproliferative properties in keloids and as a candidate therapeutic target.

Keloids are benign fibroproliferative skin tumors caused by aberrant wound healing that can negatively impact patient quality of life. The lack of animal models has limited research on pathogenesis or developing effective treatments, and the etiology of keloids remains unknown. Here, we found that the characteristics of stem-like cells from keloid lesions and the surrounding dermis differ from those of normal skin. Furthermore, the HEDGEHOG (HH) signal and its downstream transcription factor GLI1 were upregulated in keloid patient-derived stem-like cells. Inhibition of the HH-GLI1 pathway reduced the expression of genes involved in keloids and fibrosis-inducing cytokines, including osteopontin. Moreover, the HH signal inhibitor vismodegib reduced keloid reconstituted tumor size and keloid-related gene expression in nude mice and the collagen bundle and expression of cytokines characteristic for keloids in ex vivo culture of keloid tissues. These results implicate the HH-GLI1 pathway in keloid pathogenesis and suggest therapeutic targets of keloids.

Examination of Epithelial Mesenchymal Transition in Keloid Tissues and Possibility of Keloid Therapy Target.

BACKGROUND: Keloid is a fibroproliferative skin disorder that is characterized by collagen accumulation and blood vessel proliferation in the reticular layer of the dermis. It is caused by prolonged inflammation after cutaneous injury. Several studies suggested recently that epithelial mesenchymal transition (EMT) is involved in the development of fibrosis. This study assessed whether EMT also participates in keloid development and/or aggravation. METHODS: Resected keloid (n = 19) and normal skin (n = 13) samples were subjected to immunohistochemical, immunofluorescent, and Western blot analyses of their expression of epidermal (E-cadherin) and mesenchymal (vimentin) proteins. RESULTS: Immunohistochemical analysis showed that the keloid tissues had more vimentin-positive cells in the epidermis than the normal tissues. When normal primary keratinocytes were cultured with proinflammatory cytokines, the cobblestone-shaped cells changed to a spindle shape and many vimentin-positive cells were detected. When immortalized HaCaT keratinocytes were cocultured in split-well plates with normal or keloid-derived fibroblasts, they also underwent EMT, as indicated by their greater vimentin expression on Western blot analysis compared with HaCaT cells that were cultured alone. CONCLUSIONS: EMT was observed in keloid specimens. EMT was induced by inflammatory cytokines and fibroblasts. EMT may be involved in keloid generation and/or aggravation and may have potential as a keloid treatment target.

Different Patterns of Acetylation and Dimethylation of Histone H3 between Young and Aged Cases with Chronic Tonsillitis: Influences of Inflammation and Aging.

INTRODUCTION: Epigenetics is now considered to be crucially involved in normal genetics and differentiation and in pathological conditions, such as cancer, aging, and inflammation. Epigenetic mechanisms involve DNA methylation and histone modifications. The purpose of this study was to investigate the effects of inflammation on epigenetics in young subjects and the effect of aging. MATERIALS AND METHODS: The palatine tonsils were extracted from child and adult patients with chronic tonsillitis. Hematoxylin-eosin staining was performed to examine the morphology of the palatine tonsils. A fluorescence immunological examination was also performed to detect acetyl-histone H3 or dimethyl-histone H3. Confocal scanning microscopy was used for observations. RESULTS: Acetylated histone H3 was detected in tonsils from child patients but not from adult patients. Dimethylated histone H3 was not detected in tonsils from either group of patients. Degeneration of the tonsillar structures was apparent in tonsils from adult patients. DISCUSSION: The differential expression of acetylated histone H3 Lys9 may reflect immunological differences between young and aged tonsils. The decrease observed in the activity of histone methyltransferase induced the down-regulated expression of methylated histone H3. CONCLUSION: Our results suggest that epigenetic changes participate in chronic inflammation and aging in the palatine tonsils. Although the results do not lead to a direct treatment, the epigenetic pathogenesis of chronic inflammation, such as immunoglobulin A nephropathy, by focal infections will be described in greater detail in future studies, which will lead to new treatments being developed.

Identification and characterization of Wnt signaling pathway in keloid pathogenesis.

Keloid is characterized by fibroblastic cell proliferation and abundant collagen synthesis. Numerous studies have shown that the Wingless type (Wnt) signaling pathways play key roles in various cellular functions including proliferation, differentiation, survival, apoptosis and migration. The aim of this study was to clarify the role of Wnt signaling pathway in keloid pathogenesis. Primary fibroblast cultures and tissue samples from keloid and normal appearing dermis were used. The expression of Wnt family members, frizzled (FZD)4 receptor, receptor tyrosine kinase-like orphan receptor (ROR)2 and the Wnt signaling downstream targets, glycogen synthase kinase (GSK)3-ß and ß-catenin were assessed using semi-quantitative RT-PCR, Western blot, or immunohistochemical methods. Of the Wnt family members, Wnt5a mRNA and protein levels were elevated in keloid fibroblasts (KF) as compared to normal fibroblasts (NF). A higher expression of ß-catenin protein was also found in KF. No detectable levels of FZD4 receptor and ROR2 proteins were observed in both NF and KF. Functional analysis showed that treatment of NF and KF with recombinant Wnt5a peptide resulted in an increase in protein levels of total ß-catenin and phosphorylated ß-catenin at Ser33/37/Thr 41 but no significant change in phosphorylated ß-catenin at Ser45/Thr 41 positions. In addition, the expression of total GSK3-ß protein was not affected but its phosphorylated/inactivated form was increased in NF and KF. Our findings highlight a potential role for a Wnt/ß-catenin canonical signaling pathway triggered by Wnt5a in keloid pathogenesis thereby providing a new molecular target for therapeutic modulations.

Sunitinib, a small-molecule receptor tyrosine kinase inhibitor, suppresses neointimal hyperplasia in balloon-injured rat carotid artery.

The migration and proliferation of vascular smooth muscle cells (VSMCs) induced by growth factors play a critical role in in-stent stenosis after percutaneous coronary intervention (PCI). The present study tested the hypothesis that sunitinib malate (sunitinib), a tyrosine kinase inhibitor of multiple receptors for growth factors, can reduce neointimal formation after arterial injury in vivo and sought to reveal the underlying mechanism in vitro. Male Wistar rats with balloon-injured carotid arteries were administered either sunitinib or a vehicle orally for 2 weeks. Sunitinib significantly inhibited neointimal hyperplasia relative to control by reducing active cell proliferation. In cultured human aortic smooth muscle cells (HASMCs), sunitinib significantly inhibited platelet-derived growth factor (PDGF)-induced increases of DNA synthesis, cell proliferation, and migration relative to controls as evaluated by [(3)H] thymidine incorporation, cell number, and the Boyden chamber assay, respectively. Immunoblot analyses showed that sunitinib suppressed phosphorylation of PDGF-BB inducible extracellular signal-regulated kinase and autophosphorylation of PDGF ß-receptor, which are the key signaling steps involved in HASMC activation. These results indicate that sunitinib inhibits neointimal formation after arterial injury by suppressing VSMC proliferation and migration presumably through inactivation of PDGF signaling. As such, it may be a potential therapeutic agent, which targets arterial restenosis after PCI.

Evaluation of fractional analysis of bronchoalveolar lavage combined with cellular morphological features.

BACKGROUND: The value of bronchoalveolar lavage (BAL) still remains controversial, prompting a need for further improvement. The purpose of this study was to develop and evaluate a sequential analysis of cell content in fractional BAL (FBAL) from the airways and alveolar sacs with incorporation of the cellular morphologic features. METHODS: Initially, 30 ml saline was infused into a subsegmental lobe of the lung and the recovered fluid was assigned as FBAL-I being mainly originated from whole airways. The second and third lavages (FBAL-II and FBAL-III) each were performed using 50 ml saline being from more distal portions of airways and alveolar sacs respectively in the same lobe. Total cell number/ml and percentages of macrophages, lymphocytes, neutrophils, and eosinophils in each fraction together with their morphological alterations and mast cells, basophils and Masson bodies were assessed. RESULTS: In the 12 controls, percentage of neutrophils was high and lymphocytes and macrophages were low in FBAL-I while in FBAL-III, neutrophils decreased to nearly nil and lymphocytes and macrophages were increased. Analysis of FBAL from 76 patients with sarcoidosis and 14 with hypersensitivity pneumonitis (HP) revealed that a predominance of small, round and well-differentiated lymphocytes with relative absence of neutrophils, basophils and Masson bodies correlated best with sarcoidosis. In contrast, neutrophil predominance and presence of lymphocytes having deep nuclear indentations and abundant cytoplasm with a process resembling a "hand-mirror" correlated well with HP. CONCLUSIONS: Evaluation of FBAL together with cellular morphological features especially characteristics of lymphocytes provides valuable information for establishing the diagnosis in interstitial lung diseases.

A novel technique for observing the internal ultrastructure of human chromosomes with known karyotype.

Observation of the internal ultrastructure of human chromosomes by transmission electron microscopy (TEM) has frequently been attempted in spite of the difficulties in detaching metaphase chromosome spreads from the glass slide for further processing. In this study we have used a method in which metaphase chromosome spreads were prepared on a flexible thermoplastic membrane (ACLAR) film. To assess chromosome identity, a diamidino-phenylindole staining and karyotying was first done using a conventional cytogenetic system. The chromosome spreads were then fixed with 1% osmium tetroxide, stained with freshly prepared 2% tannic acid, dehydrated, and flat-embedded in epoxy resin. The resin sheet was easily detachable and carried whole chromosome spreads. By this method, TEM observation of chromosomes from normal human lymphocytes allowed a thorough examination of the ultrastructure of centromeres, telomeres, fragile sites, and other chromosomal regions. Various ultrastructural patterns including thick electron dense boundaries, less dense internal regions, and extended chromatin loops at the periphery of the chromosomes were discernible. Application of the present method to chromosome research is expected to provide comprehensive information on the internal ultrastructure of different chromosomal regions in relation to function.

Angiogenic switching in the alveolar capillaries in primary lung adenocarcinoma and squamous cell carcinoma.

The status of angiogenic switching was examined in alveolar capillaries of primary lung adenocarcinoma (ADC) from 10 patients and primary squamous cell carcinoma (SCC) from 11 patients, using immunostaining for CD31, thrombomodulin, von Willebrand factor (vWF), collagen types IV and VII, and alpha-smooth muscle actin (alpha-SMA). We applied the TdT-mediated dUTP nick-end labeling assay and the reverse transcription-polymerase chain reaction for vascular endothelial growth factor (VEGF) and its receptors (VEGFRs). In bronchioloalveolar and papillary subtypes of ADC, the neoplastic cells, replacing the normal alveolar epithelial cells, had spread over alveolar walls and adhered firmly to alveolar interstitium as shown by the development of type IV collagen. Neoplastic cells of SCC were characterized by local proliferation in alveolar sacs without firm attachment to alveolar walls. Tumor lesions of SCC had often developed necrotic foci of various size. In ADC and SCC, alveolar capillary endothelial cells newly obtained reactivity to vWF. Such segments of endothelial cells lost surface thrombomodulin expression. CD31 was consistently expressed in normal and ADC tissues, but each endothelial cell marker was often attenuated or even lost in SCC, suggesting degeneration or necrosis of the alveolar capillaries. The capillary pericytes and interstitial fibroblasts were often hypertrophic and developed alpha-SMA in the cytoplasm in ADC, but they became atrophic in SCC. In ADC, apoptosis occurred in cells of alveolar capillaries more frequently in the peripheral zone than in the deeper zone of the tumor, whereas the frequency was not consistent in SCC. In microdissected alveolar wall tissues, mRNA expression patterns of VEGF isoforms and VEGFRs were similar in both ADC and SCC. In ADC, de novo angiogenic switching took place in cytoplasm as a unit of cells segments in alveolar capillary endothelium. Suppression of angiogenic switching in SCC implies that factors other than VEGF-VEGFR interaction, such as physical contact and compression of tumor cells, might play a critical role in alveolar capillaries.

Effect of ionizing irradiation on human esophageal cancer cell lines by cDNA microarray gene expression analysis.

To provide new insights into the molecular mechanisms underlying the effect of irradiation on esophageal squamous cell carcinomas (ESCCs), we used a cDNA microarray screening of more than 4,000 genes with known functions to identify genes involved in the early response to ionizing irradiation. Two human ESCC cell lines, one each of well (TE-1) and poorly (TE-2) differentiated phenotypes were screened. Subconfluent cells of each phenotype were treated with single doses of 2.0 Gy or 8.0 Gy irradiations. After a 15 min incubation time-point, the cells were collected and analyzed. Compared with non-irradiated cells, many genes revealed at least 2-fold upregulation or downregulation at both doses in well or poorly differentiated ESCC cells. The common upregulated genes in well and poorly differentiated cell types at both irradiation doses included SCYA5, CYP51, SMARCD2, COX6C, MAPK8, FOS, UBE2M, RPL6, PDGFRL, TRAF2, TNFAIP6, ITGB4, GSTM3, and SP3 and common downregulated genes involved NFIL3, SMARCA2, CAPZA1, MetAP2, CITED2, DAP3, MGAT2, ATRX, CIAO1, and STAT6. Several of these genes were novel and not previously known to be associated with irradiation. Functional annotations of the modulated genes suggested that at the molecular level, irradiation appears to induce a regularizing balance in ESCC cell function. The genes modulated in the early response to irradiation may be useful in our understanding of the molecular basis of radiotherapy and in developing strategies to augment its effect or establish novel less hazardous alternative adjuvant therapies.