Developmental Anomalies in Human Teeth: Odontoblastic Differentiation in Hamartomatous Calcifying Hyperplastic Dental Follicles Presenting with DSP, Nestin, and HES1.

Hyperplastic dental follicles (HDFs) represent odontogenic hamartomatous lesions originating from the pericoronal tissues and are often associated with impacted or embedded teeth. These lesions may occasionally feature unique calcifying bodies, known as calcifying whorled nodules (CWNs), characterized by stromal cells arranged in a whorled or spiral fashion. CWNs are typically observed in multiple calcifying hyperplastic dental follicles or regional odontodysplasia. In our study, we examined 40 cases of HDFs, including nine instances with characteristics of CWNs, referred to as calcifying hyperplastic dental follicles (CHDFs), which are infrequently accompanied by odontodysplasia. The median ages of the HDFs and CHDFs were 16 (ranging from 3 to 66) and 15 (ranging from 11 to 50) years, respectively. The lower third molars were the most frequently affected by HDSFs and CHDFs, followed by the upper canines. A histological examination was conducted on all 40 cases, with an immunohistochemical analysis performed on 21 of them. Among the cases with CWN, nine affected a single embedded tooth, with one exception. CWNs exhibited diverse calcifications featuring sparse or entirely deposited psammoma bodies, and some displayed dentinoid formation. Immunohistochemically, the stromal cells of HDFs were frequently positive for CD56 and nestin. By contrast, CWNs were negative for CD56 but positive for nestin as well as hairy and enhancer split 1 (HES1), with a few dentin sialoprotein (DSP)-positive calcified bodies. Our results revealed that hamartomatous CHDFs can impact multiple and single-embedded teeth. CWNs composed of nestin and HES1-positive ectomesenchymal cells demonstrated the potential to differentiate into odontoblasts and contribute to dentin matrix formation under the influence of HES1. This study is the first report documenting odontoblastic differentiation in HDFs. The rare occurrence of HDFs and CHDFs contributes to limited comprehension. To prevent misdiagnosis, a better understanding of these conditions is necessary.

Sclerosing Odontogenic Carcinoma With a Prominent Clear Cell Component Mimicking Odontogenic Clear Cell Carcinoma: An Extremely Rare Case With a Fatal Clinical Outcome.

Sclerosing odontogenic carcinoma (SOC) is an exceedingly rare odontogenic carcinoma known for its locally aggressive yet indolent behavior. There have been no reports of metastasis to distant organs, except a single case involving lymph node metastasis. This report details the case of a 49-year-old female who presented with a well-demarcated radiolucent lesion in the mandible, accompanied by root resorption and tooth displacement. Microscopically, the lesion exhibited a distinctive composition, with two distinct components: cords of epithelium embedded within an abundant collagenous stroma and solid nests of clear polygonal cells surrounded by hyalinized stroma. Notably, the tumor exhibited direct invasion into the submental muscles, accompanied by perineural and vascular invasion, as well as cortical bone loss. Additionally, the clear cells contained diastase-sensitive periodic acid-Schiff-positive granules. Immunohistochemically, the tumor cells displayed positivity for cytokeratin 19 and p63 while testing negative for myoepithelial markers. The Ki-67 index was measured at 23%. Importantly, neitherEWSR1 nor MAML2 rearrangements were detected through fluorescence in situ hybridization (FISH) analysis. Over several years, this patient experienced three instances of local recurrence; notably, four years after the initial surgery, fludeoxyglucose F18-positron emission tomography (18FDG-PET)/CT scans confirmed the presence of pulmonary metastasis. This case presents an unusual histological variation of SOC, marked by vascular invasion, and is notably the first documented case of a fatal outcome in this context.

Comprehensive cornified envelope protein profile of odontogenic keratocysts clarifies the characteristics of non-keratinized oral epithelium.

BACKGROUND: Odontogenic keratocysts constitute 10%-20% of odontogenic cysts and exhibit a distinctive corrugated parakeratinized lining epithelium. Considering that cornified envelope formation is an important phenomenon during keratinocyte differentiation, this study aimed to clarify the characteristics of cornified envelope formation in odontogenic keratocysts. METHODS: We investigated the cellular distribution of cornified envelope-related proteins (transglutaminases and their substrates), as well as the upstream regulatory protein c-Fos, by immunohistochemical analysis of the lining epithelium of 20 odontogenic keratocysts. We examined the corresponding mRNA levels by quantitative polymerase chain reaction. Ten dentigerous cysts served as control non-keratinized cysts. RESULTS: The distributions of transglutaminase and their substrates except loricrin and small protein-rich protein 1a significantly differed between odontogenic keratocysts and dentigerous cysts. There was no significant difference in c-Fos expression between odontogenic keratocysts and dentigerous cysts. The mRNA levels of transglutaminases and their substrates were significantly higher in odontogenic keratocysts than in dentigerous cysts. However, c-Fos mRNA levels did not significantly differ between groups. CONCLUSION: Surprisingly, the overall appearance of cornified envelope-related proteins of odontogenic keratocysts was consistent with the characteristics of non-keratinized oral mucosa identified in previous studies. These findings indicate that the contribution of cornified envelope-related molecules in odontogenic keratocysts is similar to that in non-keratinized oral epithelium, rather than keratinized oral epithelium, suggesting that odontogenic keratocysts are not genuine keratinized cysts. The upregulation of cornified envelope-related genes in odontogenic epithelium could be an important pathognomonic event during odontogenic keratocyst development.

Carcinoma Cuniculatum of the Maxilla Arising From Oroantral Fistula: A Report of an Extremely Rare Case.

Carcinoma cuniculatum (CC) is extremely rare in the maxilla. Here, we report a case of CC arising from an oroantral fistula (OAF). The patient was a 70-year-old Japanese man who was followed up for a non-closing OAF. Although there were no findings based on an intraoral examination, follow-up contrast-enhanced computed tomography and magnetic resonance imaging showed a 22-mm mass in the maxilla close to the OAF. Histologically, cystic and endophytic papillary proliferation of squamous epithelium with abundant keratinization mimicking rabbit burrows occupied the alveolar bone. This tumor was directly connected to the atypical proliferation of the covering epithelium of the OAF. The tumor cells showed mild cytological atypia and a few mitoses. Finally, the patient was diagnosed with CC arising from an OAF. CC is often misdiagnosed; nonetheless, the unique endophytic, branching, and tunnel-like structure is a hallmark of this tumor. We present the first well-documented case of CC arising from an OAF, discuss its diagnostic features, and highlight its differences from other common benign and malignant pathological entities.

Composite hemangioendothelioma in the cervical spine with kaposiform hemangioendothelioma features in an elderly patient: a case report.

BACKGROUND: Composite hemangioendothelioma (CHE) is an intermediate group of tumors with features between hemangioma and angiosarcoma both histologically and biologically. CHE is predominant in young and middle-aged adults, but very infrequently affects the spine. We describe the case of primary CHE in the cervical spine exhibiting kaposiform hemangioendothelioma (KHE)-like components that was associated with cervical myelopathy with vertebral body destruction in an elderly woman. We retrospectively reviewed the case of a primary cervical spinal tumor, diagnosed as CHE with KHE-like components in pathological findings, associated with cervical myelopathy and cervical vertebral body destruction. CASE PRESENTATION: An 80-year-old woman presented with progressive cervical myelopathy caused by a cervical spine tumor. Preoperative cervical MRI revealed a neoplastic lesion invading the cervical spine that strongly compressed the spinal cord, causing right upper-limb paralysis. We performed partial tumor resection along with posterior decompression and fixation. Postoperatively, pathological findings showed that the tumor was CHE with KHE-like features. Following radiotherapy, no recurrences have been observed in 21 months. CONCLUSIONS: This is the first report of CHE with features of KHE in the spine of an elderly patient. Posterior decompression and fusion of the cervical spine and subsequent radiotherapy resulted in a good outcome.

A Case of Oral Glomeruloid Hemangioma Without Systemic Conditions.

Glomeruloid hemangioma is a rare variant of hemangioma that is accompanied by polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin abnormalities (POEMS) syndrome and, rarely, by thrombocytopenia, anasarca, fever, reticulin fibrosis, and organomegaly (TAFRO) syndrome. This report presents the case of a 78-year-old male who presented with a hemorrhagic nodule on the tongue without any other systemic diseases. Microscopically, the lesion was a lobular proliferation extending from the lamina propria to muscular tissue. Some intravascular nodules with irregular vascular lumens closely resembled renal glomeruli. Each nodule consisted of plump endothelial and stromal cells that partially showed vacuolated cytoplasm containing eosinophilic and periodic acid-Schiff (PAS)-positive globules. Immunohistochemically, IgG-positive deposition was noted within CD31-positive cells. Many plump stromal cells were positive for CD31, CD146, nestin, and type IV collagen but not α-smooth muscle actin (αSMA). These results reflect the proliferation of immature endothelial cells and pericytes, which might characterize this unique lesion. Microscopically, this case revealed glomeruloid hemangioma without systemic conditions related to POEMS, and composed of an intravascular proliferation of immature endothelial and pericytic stromal cells.

Mammaglobin protein localization and gene expression in the salivary glands.

PURPOSE: This study aims to delve deeper into the hypothesis that normal salivary gland tissue expresses both protein and mRNA of mammaglobin (MGB). METHODS: Formalin-fixed paraffin-embedded samples of submandibular (10), parotid (5), palatal (5) and labial glands (30) salivary glands were immunohistochemically investigated. The labial samples were used to examine the MGB positive ratio (MGB-PR), and localize MGB by double immunofluorescence staining and quantitative mRNA gene expression. Mann-Whitney U and Kruskal Wallis rank-sum test for group comparison, and Spearman's rank correlation coefficient for correlation analysis were used. RESULTS: The distribution of MGB-positive cells was variable throughout samples with significantly higher MGB-PR of acini than ducts (P = 0.00376), and there was no difference when compared based on age (P = 0.0646) and gender (P = 0.245). Besides acinar cells, a number of myoepithelial cells and ductal cells also demonstrated strong MGB reactivity with varying MGB mRNA expression levels in 6 of the 7 samples (with MGB-PR > 20%) tested. CONCLUSION: This novel study shows that unlike aberrant protein expression in some carcinomas, MGB expression in salivary gland neoplasms represents the nature of original cells, giving a better insight into the neoplasms expressing MGB.

Contribution of transglutaminases and their substrate proteins to the formation of cornified cell envelope in oral mucosal epithelium.

Cornified envelope formation is crucial for the final differentiation of keratinized epithelium. However, the mechanisms of cornified envelope formation in the oral epithelium remain unclear. The aim of this study was to clarify the differences in the distribution and expression of cornified envelope related proteins and genes between keratinized and non-keratinized oral epithelia. We immunohistochemically investigated the distribution patterns of transglutaminase 1 (TG1), transglutaminase 3 (TG3), and their substrate proteins involucrin (IVL), loricrin (LOR), and small proline rich proteins (SPRs), in 19 keratinized and 14 non-keratinized oral epithelium samples. TG1 and TG3 mRNA levels were investigated in both types of epithelium by real time reverse transcription polymerase chain reaction (RT-PCR) using paraffin-embedded specimens. Data were analyzed to identify factors involved in cornified envelope formation. We demonstrate that 11 localization patterns show statistically significant differences between keratinized and non-keratinized oral epithelia. These factors clearly drove the separation of the two groups during cluster analysis. TG1 mRNA levels in keratinized oral epithelium were significantly higher than those in non-keratinized oral epithelium. In conclusion, the characteristic distribution of transglutaminases and their substrates and the mRNA levels of TG1 can regulate cornified envelope formation in keratinized oral epithelium, together with the contribution of TG3 first reported in this paper.

Lymphoepithelioma-like cholangiocarcinoma with Epstein-Barr virus infection treated by radiofrequency ablation.

Lymphoepithelioma-like cholangiocarcinoma (LELCC) is a rare intrahepatic tumor. There are usually no specific physical findings, and the tumors are often diagnosed incidentally and are frequently large-sized at diagnosis. The imaging findings of LELCC resemble those of hepatocellular carcinoma (HCC). Tumors are often found in large-sized and advanced at diagnosis, and the main treatment of the disease is surgical resection. Herein, we report treating a patient with early stage LELCC by radiofrequency ablation (RFA). We diagnosed this tumor in a 27-year-old Chinese female with a history of chronic hepatitis B (CHB). Based on the findings of blood examination, abdominal ultrasonography, and gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI), this tumor was diagnosed as suspected HCC. Ultrasound-guided percutaneous tumor biopsy and RFA were performed at the same time. The histopathological findings finally revealed the diagnosis of LELCC. To the best of our knowledge, this is the first report, in the English-language literature, of the treatment of LELCC by RFA; we suggest that RFA might be a candidate treatment for small-sized early stage LELCC.

Homeobox transcription factor engrailed homeobox 1 is a possible diagnostic marker for adenoid cystic carcinoma and polymorphous adenocarcinoma.

Diagnostic utility of a homeobox transcription factor, engrailed homeobox 1 (En1) in the histopathology of salivary gland neoplasms was studied. The expression of En1 was immunohistochemically examined in 51 cases of adenoid cystic carcinoma (AdCC) and 143 cases of other salivary gland neoplasms. In all 51 AdCCs, En1 was expressed in 30-100% of tumor cells. In eight of nine polymorphous adenocarcinomas (PACs), En1 was expressed in 40-100% of tumor cells. Less than 5% of tumor cells expressed En1 in three of 12 epithelial-myoepithelial carcinomas, one of 17 basal cell adenomas (BCAs), and one of 34 pleomorphic adenomas (PAs). Among 55 other carcinoma cases, 1-30% of tumor cells expressed En1 in three salivary duct carcinomas (SDCs) ex PA. None of the myoepitheliomas and Warthin tumors expressed En1. When the cut-off value of the percentage of En1-expressing cells was set to 25%, all 51 AdCCs, eight of nine PACs and one SDC ex PA were En1-positive and the others were En1-negative. En1 is expressed consistently in AdCCs, frequently in PACs, but rarely in other salivary gland neoplasms. En1 is a possible diagnostic marker for AdCC and PAC in the histopathology of salivary gland neoplasms.

Pharyngeal Deposits Comprising Salivary Mucin in Tube-fed Elderly Patients: MUC2 and MUC7 Immunoreactivity.

Several investigators have reported that oral membranous and pharyngeal viscous deposits developed in bedridden elderly persons requiring nursing care without oral intake. Therefore, this study aimed to clarify the origin of viscous deposits on the pharyngeal mucosa based on characteristics of salivary and tracheal secretory mucin. The participants were 35 elderly people who required nursing care. All 46 collected specimens, including 30 intraoral and 16 pharyngeal specimens, were stained against specific mucins secreted from the respiratory tract and saliva gland using antibodies anti-MUC2 and anti-MUC7, respectively. Out of 35 participants, the intraoral membranous deposits and deposits on the pharyngeal mucosa developed in 17 (48.6%) and 10 persons (28.6%), respectively. The pharyngeal deposits developed in 58.8% of participants who developed intraoral deposits. All pathological specimens shared microscopic findings of various combinations of eosinophilic lamellar structure and a pale-basophilic amorphous substance. Immunohistochemically, both the 30 oral and the 16 pharyngeal specimens obtained from 17 participants were consistently positive for MUC7 but negative for MUC2. In conclusion, we clarified that the mucoid component of both oral and pharyngeal deposits comprised MUC7 salivary mucin, which revealed that both deposits originated from the oral cavity. This result strongly suggests that oral care is intimately related to oral and pharyngeal conditions.

Histopathological evaluation of oral membranous substance in bedridden elderly persons without oral intake in Japan.

OBJECTIVES: The aim of this study was to clarify by histopathological examination the origin of oral membranous substances deposited on the palate, tongue, buccal mucosa and teeth. BACKGROUND: Several investigators have reported membranous substances deposited in the mouths of bedridden elderly persons requiring nursing care without oral intake. However, the precise nature and origin of the substances are poorly understood. METHODS: Sixty-nine specimens were taken from the oral cavity of bedridden patients, that is, the palate, dorsum of the tongue, the cheek and teeth. Sections were stained with haematoxylin and eosin stain, alcian-blue and periodic acid-Schiff stain (AB-PAS) and antibodies for pankeratin (AE1AE3) and leukocyte common antigen (LCA). RESULTS: All specimens showed a film-like nature coloured from tan to white, accompanied by a mucous substance. Histologically, specimens of all sites had a similar feature of the combination of basophilic amorphous and eosinophilic lamellar features. The basophilic substance was positive for AB-PAS, and PAS-positive glycogen granules were also noted in the lamellar structure. Immunochemistry revealed various degrees of pankeratin positive substance and LCA-positive inflammatory cell infiltration. CONCLUSION: The oral membranous substance was composed of keratin and mucin with inflammation. These results suggest that the deposition of the oral membranous substance is a pathological condition or oral mucositis caused by dry mouth.

Ectopic transglutaminase 1 and 3 expression accelerating keratinization in oral lichen planus.

OBJECTIVE: Oral lichen planus (OLP) characterized by interface mucositis frequently shows hyper-keratinization. To clarify mechanisms of excess keratinization, we investigated key molecules for cornified cell envelope (CE). METHODS: Involucrin (IVL), loricrin (LOR), transglutaminase 1 (TGase 1) and transglutaminase 3 (TGase 3) were immunohistochemically examined in 20 specimens of OLP; five specimens of buccal mucosa served as controls. Subsequently, the data were statistically analyzed. RESULTS: IVL in OLP was localized in the cell membrane, in contrast to its localization in the cytoplasm in controls. No positive reaction indicative of LOR was noted in any specimens. Although the TGase 1 localization in controls was restricted to the upper three-quarters of the membrane, the localization in OLP was in both membrane and in the cytoplasm of full thickness mucosal layers. The TGase 3 localization pattern was dramatically altered from cytoplasmic to membranous in OLP. CONCLUSION: Our data suggest that aberrant TGase 1 and TGase 3 localization and distribution are closely related to hyper-keratinization in OLP. This is the first report of ectopic transglutaminase localization in OLP.

Phenotypic alteration of basal cells in oral lichen planus; switching keratin 19 and desmoglein 1 expression.

In oral lichen planus, extracellular matrix and basal cells are damaged by T-lymphocytes. As a consequence, changes in expression of collagen fibers within the connective tissue and cytoskeletons of the epithelial tissue can be observed. With the goal of examining the characteristic changes undergone by basal cells as a consequence of T-lymphocytes damage in oral lichen planus, we investigated protein expression in the epithelial-connective junction. We selected 20 cases of oral lichen planus and 5 control samples of buccal mucosa. Subsequently, we divided the oral lichen planus cases into thin and thick parts based on the mean values of epithelial thickness from the control samples, and counted the positive rate of collagen IV, keratin 19, desmoglein 1, and Ki-67. Collagen IV immune-reactivity partially disappeared or thickened in oral lichen planus. The keratin 19 positive rate in oral lichen planus cases was significantly lower than in the controls. Desmoglein 1 positive rate of the thick part was significantly higher compared to the thin part of oral lichen planus. Thus, modifications in basal cells with both reduced keratin 19 expression and alterations of desmoglein 1 expression suggest that in oral lichen planus, as a consequence of cell injury or regeneration in the interface area, there is a disappearance of the "true basal cell nature".

Cell division cycle 34 is highly expressed in hepatitis C virus-positive hepatocellular carcinoma with favorable phenotypes.

Despite tremendous efforts to develop curative agents, there are few effective drugs for the treatment of hepatocellular carcinoma (HCC). This is predominantly due to the variations in individual HCC cases. As numerous HCC cases have no mutations in known tumor-associated genes, identification of novel genes involved in the development and progression of human cancers is considered to be an urgent issue. In the present study, surgical specimens of HCC were analyzed for the expression patterns of ubiquitin-conjugating enzyme, cell division cycle 34 (CDC34), which is hypomethylated in its promoter region and exhibits elevated expression levels in mouse skin tumors. The results of the current study clearly indicated that the elevated CDC34 expression level in cancerous regions was significantly associated with favorable clinicopathological features, such as reduced alanine aminotransferase (ALT) levels and histological grades. Similarly, a higher T/N ratio, which is the ratio of CDC34 expression in HCCs to that in non-tumorous tissues, was significantly associated with favorable features, such as a lower indocyanin green retention rate after 15 min (ICG15R), reduced α-fetoprotein and smaller tumor size. These results indicate that the CDC34 expression level in HCC is a marker for predicting the HCC prognosis and that CDC34 acts as a tumor suppressor.

Onset of Tuberculosis from a Pulmonary Latent Tuberculosis Infection during Antiviral Triple Therapy for Chronic Hepatitis C.

A 62-year-old man was diagnosed with the onset of tuberculosis (Tb) from a pulmonary latent tuberculosis infection (LTBI) during triple therapy with pegylated interferon α2a, ribavirin, and telaprevir for a chronic hepatitis C infection in 2013 before interferon (IFN)-free anti-viral therapy was introduced in Japan. A liver biopsy before IFN treatment revealed the presence of epithelioid cell granulomas (ECGs). IFN may also be employed for chronic hepatitis B infection and malignant tumors, thus, special attention must be paid to the development of Tb from a LTBI when ECGs are observed before treatment. It is also necessary to review the significance of the liver biopsy.

Migration and Differentiation of GFP-transplanted Bone Marrow-derived Cells into Experimentally Induced Periodontal Polyp in Mice.

Perforation of floor of the dental pulp is often encountered during root canal treatment in routine clinical practice of dental caries. If perforation were large, granulation tissue would grow to form periodontal polyp. Granulation tissue consists of proliferating cells however their origin is not clear. It was shown that the cells in granulation tissue are mainly from migration of undifferentiated mesenchymal cells of the bone marrow. Hence, this study utilized GFP bone marrow transplantation mouse model. The floor of the pulp chamber in maxillary first molar was perforated using ½ dental round bur. Morphological assessment was carried out by micro CT and microscopy and GFP cell mechanism was further assessed by immunohistochemistry using double fluorescent staining with GFP-S100A4; GFP-Runx2 and GFP-CD31. Results of micro CT revealed alveolar bone resorption and widening of periodontal ligament. Histopathological examination showed proliferation of fibroblasts with some round cells and blood vessels in the granulation tissue. At 2 weeks, the outermost layer of the granulation tissue was lined by squamous cells with distinct intercellular bridges. At 4 weeks, the granulation tissue became larger than the perforation and the outermost layer was lined by relatively typical stratified squamous epithelium. Double immunofluorescent staining of GFP and Runx2 revealed that both proteins were expressed in spindle-shaped cells. Double immunofluorescent staining of GFP and CD31 revealed that both proteins were expressed in vascular endothelial cells in morphologically distinct vessels. The results suggest that fibroblasts, periodontal ligament fibroblasts and blood vessels in granulation tissue were derived from transplanted-bone marrow cells. Thus, essential growth of granulation tissue in periodontal polyp was caused by the migration of undifferentiated mesenchymal cells derived from bone marrow, which differentiated into fibroblasts and later on differentiated into other cells in response to injury.

The role of macrophage transcription factor MafB in atherosclerotic plaque stability.

BACKGROUND AND AIMS: Macrophage differentiation is associated with the development of atherosclerosis and plaque vulnerability and is regulated by transcription factor MafB. We previously reported that MafB attenuates macrophage apoptosis, which is associated with atherosclerotic plaque instability. The aim of this study was to elucidate the role of MafB in the progression of atherosclerotic plaque. METHODS: We generated macrophage-specific dominant-negative (DN) MafB transgenic mice and intercrossed DN-MafB mice with apolipoprotein E (ApoE) knockout (KO) mice. RESULTS: There was no significant difference in advanced atherosclerotic lesion area between DN-MafB/ApoE KO mice and littermate control ApoE KO mice 9 weeks after high-cholesterol diet. However, DN-MafB/ApoE KO mice showed significantly larger necrotic cores and lower collagen content in atherosclerotic plaques than ApoE KO mice. Although there was no difference in intraplaque macrophage infiltration and efferocytosis, DN-MafB/ApoE KO mice showed significantly more apoptotic macrophages at the plaque edges than did ApoE KO mice. Real-time PCR analysis revealed that peritoneal macrophages of DN-MafB/ApoE KO mice had a greater increase in matrix metalloproteinase-9 and mRNA expression of inflammatory/M1 macrophage markers (tissue necrosis factor-α, interleukin-6, CD11c, and p47phox) after lipopolysaccharide stimulation than those of ApoE KO mice. CONCLUSION: Macrophage-specific inhibition of MafB may destabilize atherosclerotic plaques in advanced lesions.

Pathological Analysis of Cell Differentiation in Cholesterol Granulomas Experimentally Induced in Mice.

In this study, cholesterin was implanted in the subcutaneous tissue in mice to induce the formation of cholesterol granuloma. Histological examination was carried out to determine the type and source of cells. The tissue surrounding the embedded cholesterin was examined histologically within the period of 6 months. Cell differentiation in cholesterol granulomas was investigated using ddY mice and GFP bone marrow transplanted mice. Cholesterin was embedded in mice subcutaneously and histopathological examination was carried out in a period of 6 months. Results showed that at 2 weeks, cholesterin was replaced partly by granulation tissues. The majority of cells in the granulation tissues were macrophages and foreign body giant cells and the center consists of small amount of fibroblasts, collagen fibers and capillaries. At 3 months, more granulation tissue was observed compared to 2 weeks. Similar cells were observed, however, there were more fibroblasts, collagen bundles and capillaries present compared to 2 weeks. At 6 months, the cholesterin was mostly substituted by fibrous tissues consisting mainly of fibroblasts and collagen fibers with some macrophages and foreign body giant cells. Specifically, the outer part of the tissue consists of fibroblasts, collagen bundles and capillaries and the inner portion is filled with collagen bundles. Immunohistochemistry revealed that macrophages and foreign body giant cells were positive to GFP and CD68 although the fibroblasts and capillaries in the outer portion of cholesterol granulomas were GFP negative. Some spindle shape fibroblasts were also GFP positive. Immunofluorescent double staining revealed that cells lining the blood vessels were both positive to GFP and CD31 indicating that those were endothelial cells and were actually derived from the transplanted bone marrow cells. The results suggest that macrophages, foreign body giant cells as well as fibroblasts and capillary endothelial cells are bone marrow derived mesenchymal cells.

HECT-Type Ubiquitin E3 Ligase ITCH Interacts With Thioredoxin-Interacting Protein and Ameliorates Reactive Oxygen Species-Induced Cardiotoxicity.

BACKGROUND: The homologous to the E6-AP carboxyl terminus (HECT)-type ubiquitin E3 ligase ITCH is an enzyme that plays a pivotal role in posttranslational modification by ubiquitin proteasomal protein degradation. Thioredoxin-interacting protein (TXNIP) is a negative regulator of the thioredoxin system and an endogenous reactive oxygen species scavenger. In the present study, we focused on the functional role of ubiquitin E3 ligase ITCH and its interaction with TXNIP to elucidate the mechanism of cardiotoxicity induced by reactive oxygen species, such as doxorubicin and hydrogen peroxide. METHODS AND RESULTS: Protein interaction between TXNIP and ITCH in cardiomyocyte was confirmed by immunoprecipitation assays. Overexpression of ITCH increased proteasomal TXNIP degradation and augmented thioredoxin activity, leading to inhibition of reactive oxygen species generation, p38 MAPK, p53, and subsequent intrinsic pathway cardiomyocyte apoptosis in reactive oxygen species-induced cardiotoxicity. Conversely, knockdown of ITCH using small interfering RNA inhibited TXNIP degradation and resulted in a subsequent increase in cardiomyocyte apoptosis. Next, we generated a transgenic mouse with cardiac-specific overexpression of ITCH, called the ITCH-Tg mouse. The expression level of TXNIP in the myocardium in ITCH-Tg mice was significantly lower than WT littermates. In ITCH-Tg mice, cardiac dysfunction and remodeling were restored compared with WT littermates after doxorubicin injection and myocardial infarction surgery. Kaplan-Meier analysis revealed that ITCH-Tg mice had a higher survival rate than WT littermates after doxorubicin injection and myocardial infarction surgery. CONCLUSION: We demonstrated, for the first time, that ITCH targets TXNIP for ubiquitin-proteasome degradation in cardiomyocytes and ameliorates reactive oxygen species-induced cardiotoxicity through the thioredoxin system.