Hiroshi Hano

Insufficient autophagy in idiopathic pulmonary fibrosis

Autophagy, a process that helps maintain homeostatic balance between the synthesis, degradation, and recycling of organelles and proteins to meet metabolic demands, plays an important regulatory role in cellular senescence and differentiation. Here we examine the regulatory role of autophagy in idiopathic pulmonary fibrosis (IPF) pathogenesis. We test the hypothesis that epithelial cell senescence and myofibroblast differentiation are consequences of insufficient autophagy. Using biochemical evaluation of in vitro models, we find that autophagy inhibition is sufficient to induce acceleration of epithelial cell senescence and myofibroblast differentiation in lung fibroblasts. Immunohistochemical evaluation of human IPF biospecimens reveals that epithelial cells show increased cellular senescence, and both overlaying epithelial cells and fibroblasts in fibroblastic foci (FF) express both ubiquitinated proteins and p62. These findings suggest that insufficient autophagy is an underlying mechanism of both accelerated cellular senescence and myofibroblast differentiation in a cell-type-specific manner and is a promising clue for understanding the pathogenesis of IPF.

Localization of Ang-1, -2, Tie-2, and VEGF expression at endothelial-pericyte interdigitation in rat angiogenesis.

Endothelial cells and pericytes play critical role in angiogenesis, which is controlled, in part, by the angiopoietin (Ang)/Tie-2 system and vascular endothelial growth factor (VEGF). Here, we investigated Ang, Tie-2, and VEGF expression within endothelial cells and pericyte interdigitations (EPI), which consist of cytoplasmic projections of pericytes and corresponding endothelial indentations. After subcutaneous implantation of a thermoreversible gelation polymer disc in rats, the capillary density was low on day 5, increased to a peak on day 7, and then decreased on days 10–20. A small number of EPI were observed on day 5, then increased sharply to a peak on day 10, but had decreased on day 20. Light and electron microscopy immunohistochemical and RNA in situ hybridization analyses revealed that Tie-2 localized at endothelial cells, and Ang-2 localized at endothelial cells and pericytes, while Ang-1 and VEGF localized at pericytes, and Ang-1 was most intensely observed at EPI of pericytes. Conventional quantitative RT-PCR and Western blot analyses revealed that the level of Ang-1 was low on days 5–7, then increased on days 10–20, while the level of VEGF was high on days 5–10, but had decreased on day 20. The level of Ang-2 remained high and Tie-2 remained at the level of the control on days 5–20. The present study showed that the angiogenic phase might be initiated by increases in Ang-2 and VEGF, while the microvessel maturation phase might be initiated by a relative increase in Ang-1 and a decrease in VEGF. Moreover, EPI might serve as a pathway for the Ang-1/Tie-2 system, with VEGF promoting pericyte recruitment for microvascular integrity.

Enhanced CD34 expression of sinusoid-like vascular endothelial cells in hepatocellular carcinoma.

The immunohistochemlcal expression of CD34 (human hematopoletic stem cell and endothelial cell marker) and laminin were studied In chronic liver diseases and hepate cellular carcinoma (HCC) to elucidate whether their expression reflected phenotypic differences between non‐cancerous slnusoids and sinusoid‐like tumor vessels. In normal liver, hepatic sinusoids were always negative for CD34 and lami‐nin. In chronic hepatitis and cirrhosis, the two antigens were sparsely expressed in capillarized sinusoids at periportal and petinodular area. In advanced HCC, CD34 was strongly and diffusely expressed by the endothelial lining of sinusoid‐like tumor vessels. However, early‐stage HCC showed a wide spectrum of CD34 expression from negative to focal and diffuse, strongly positive staining in sinusoid‐like vessels. Laminin was strongly expressed in advanced HCC but not in early‐stage HCC. The results Indicate that the enhanced expression of CD34 by sinusoidal endothelial cells may reflect the phenotypic change of endothellal cells in chronic liver diseases and HCC, and that the expression may correlate with the processes of angiogenesis induced by hepatocarcinogenesis.

Evaluation of new monoclonal anti-MyoD1 and anti-myogenin antibodies for the diagnosis of rhabdomyosarcoma.

New monoclonal anti‐MyoD1 and anti‐myogenin antibodies were evaluated immunohistochemically to determine whether they are useful in discriminating rhabdomyosarcoma (RMS) from other soft tissue tumors in routinely processed sections. Neither MyoD1 nor myogenin was expressed in normal, mature striated muscle. In RMS, nuclear expression of MyoD1 and myogenin was found in 82 and 80% of non‐overlapping cases, respectively. MyoD1 was generally expressed in small, primitive tumor cells, and larger cells exhibiting morphological evidence of skeletal muscle differentiation failed to express positive nuclear immunostaining. Positive nuclear staining for myogenin was stronger than that for MyoD1 in cases with abundant differentiated tumor cells, but was less prominent in cases in which small, primitive tumor cells predominated. No leiomyosarcomas, Ewing’s sarcomas/peripheral primitive neuroectodermal tumors or other soft tissue tumors exhibited nuclear expression of MyoD1 or myogenin. In conclusion, both anti‐MyoD1 and anti‐myogenin antibodies are useful for diagnosing RMS and for discriminating RMS from other soft tissue tumors.

Three-dimensional observations of the human hepatic artery: (Arterial system in the liver)

BACKGROUND/AIMS We examined the three-dimensional structures of the hepatic artery. MATERIALS/METHODS A 39-year-old man who died of brain hemorrhage was autopsied. The liver was perfused with physiological saline and 20% formalin from the hepatic artery and portal vein. More than 700 serial sections were obtained from a paraffin-embedded block, and vascular reconstruction was performed under a light microscope. RESULTS The hepatic artery divides into the axial artery and the peribiliary branch given off from it. These two systems also connect to each other by a few anastomoses. The former systematically supplies arterial blood to all the parenchymal liver cells. The latter forms two layers of plexes around the bile duct. The inner capillary layer is afferent and the outer vascular layer is efferent to the bile duct. CONCLUSION To maintain constant sinusoidal blood flow, the terminal portions of the axial arteries may contract and thereby divert blood to peribiliary branches through bifurcations and anastomoses. The blood flow of the peribiliary capillary plexus may affect bile flow. The hepatic artery may act as a functional mediator between portal flow and bile excretion.

Involvement of creatine kinase B in cigarette smoke-induced bronchial epithelial cell senescence.

Cigarette smoke induces damage to proteins and organelles by oxidative stress, resulting in accelerated epithelial cell senescence in the lung, which is implicated in chronic obstructive pulmonary disease (COPD) pathogenesis. Although the detailed molecular mechanisms are not fully understood, cellular energy status is one of the most crucial determinants for cell senescence. Creatine kinase (CK) is a constitutive enzyme, playing regulatory roles in energy homeostasis of cells. Among two isozymes, brain-type CK (CKB) is the predominant CK in lung tissue. In this study, we investigated the role of CKB in cigarette smoke extract (CSE)-induced cellular senescence in human bronchial epithelial cells (HBECs). Primary HBECs and Beas2B cells were used. Protein carbonylation was evaluated as a marker of oxidative protein damage. Cellular senescence was evaluated by senescence-associated β-galactosidase staining. CKB inhibition was examined by small interfering RNA and cyclocreatine. Secretion of IL-8, a hallmark of senescence-associated secretary phenotype, was measured by ELISA. CKB expression levels were reduced in HBECs from patients with COPD compared with that of HBECs from nonsmokers. CSE induced carbonylation of CKB and subsequently decreased CKB protein levels, which was reversed by a proteasome inhibitor. CKB inhibition alone induced cell senescence, and further enhanced CSE-induced cell senescence and IL-8 secretion. CSE-induced oxidation of CKB is a trigger for proteasomal degradation. Concomitant loss of enzymatic activity regulating energy homeostasis may lead to the acceleration of bronchial epithelial cell senescence, which is implicated in the pathogenesis of COPD.

Immunohistochemical ETS-related gene detection in a Japanese prostate cancer cohort : Diagnostic use in Japanese prostate cancer patients

Chromosomal rearrangements that result in high expression levels of the ETS‐related gene (ERG) present in approximately 50% of prostate cancer (PCa) patients, making this one of the most common oncogenic alterations in PCa. However, ERG overexpression at the protein level has not been rigorously evaluated in Japanese PCa patients. In this study, we evaluated ERG expression using antibody‐based detection in 230 prostate specimens in a Japanese PCa cohort. Overall, we identified 20.1% ERG‐positive PCa cases. ERG was not detected in benign glands. The specificity of ERG staining for detecting PCa was almost 100%; all of the ERG‐positive samples were also diagnosed as PCa. The expression level of the ERG protein correlated with clinicopathological variables, including grade (P= 0.038), stage (P= 0.005), and metastatic status (P= 0.014). No correlation was observed with age (P= 0.196) or with preoperative prostate‐specific antigen level (P= 0.322). Although the frequency of ERG‐positive cases in Japanese PCa patients (20.1%) was lower than that reported in a PCa cohort in Western countries (approximately 50%), our study demonstrates that the clinical utility of ERG detection at the protein level can serve as an ancillary tool for diagnosing PCa in the Japanese population.

Ser217Leu polymorphism of the HPC2/ELAC2 gene associated with prostatic cancer risk in Japanese men

The HPC2/ELAC2 gene may be associated with hereditary/familial prostate cancer (PCa). Two common missense variants (Ser217Leu and Ala541Thr) have been reported in the gene. We performed mutational, allelotyping and expression analyses and a molecular epidemiological study to clarify the relations between this gene and prostatic diseases, including PCa and benign prostatic hyperplasia (BPH) in Japanese men. We screened for mutations in 109 patients with PCa including 11 patients from 1 hereditary and 9 familial PCa. Loss of heterozygosity and expression were analyzed. An epidemiological study was done in sporadic PCa (n=98) and BPH (n=143) using 1 novel (Ser627Leu) and 2 previously described polymorphisms of the HPC2/ELAC2 gene. Somatic or germline mutations were not confirmed in any cases of PCa. Loss of heterozygosity at 2 microsatellites, D17S1289 and D17S520, was detected in 1 of 38 and 1 of 35 cases, respectively. Expression analysis revealed decreased or absent mRNA expression in 6 of 38 tumors. Epidemiologic analysis showed that a Leu allele at codon 217 was significantly more frequent in patients with PCa than in controls (10.2% vs. 3.5%, odds ratio = 3.11; 95% confidence interval, 1.22–7.90). At codon 541, all patients with PCa or BPH and all control subjects had the Ala/Ala genotype. At codon 627, the incidence of the Leu variant was slightly, but not significantly, higher in patients with BPH than in controls (7.0% vs. 2.8%, odds ratio = 2.59, 95% confidence interval, 0.94–7.13, not statistically significant). We concluded that germline/somatic mutations of HPC2/ELAC2 are uncommon in PCa. Similarly, allelic imbalances at the gene locus and changes in expression are rare. Although no difference in allele frequency at Ser217Leu between patients with PCa and controls has been reported in a Western population, this polymorphism is a potential indicator of PCa risk in Japanese men and it should be examined in other ethnic groups.

IQGAP1 and vimentin are key regulator genes in naturally occurring hepatotumorigenesis induced by oxidative stress

To identify key genes involved in the complex multistep process of hepatotumorigenesis, we reduced multivariate clinicopathological variables by using the Long-Evans Cinnamon rat, a model with naturally occurring and oxidative stress-induced hepatotumorigenesis. Gene expression patterns were analyzed serially by profiling liver tissues from rats of a naive status (4 weeks old), through to those with chronic hepatitis (26 and 39 weeks old) to tumor development (67 weeks old). Of 31 099 probe sets used for microarray analysis, 87 were identified as being upregulated in a stepwise manner during disease progression and tumor development. Quantitative real-time reverse transcription-polymerase chain reaction and statistical analyses verified that IQGAP1 and vimentin mRNA expression levels increased significantly throughout hepatotumorigenesis. A hierarchical clustering algorithm showed both genes clustered together and in the same cluster group. Immunohistochemical and western blot analyses showed similar increases in protein levels of IAGAP1 and vimentin. Finally, pathway analyses using text-mining technology with more comprehensive and recent gene-gene interaction data identified IQGAP1 and vimentin as important nodes in underlying gene regulatory networks. These findings enhance our understanding of the multistep hepatotumorigenesis and identification of target molecules for novel treatments.

Lecithin: retinol acyltransferase protein is distributed in both hepatic stellate cells and endothelial cells of normal rodent and human liver.

Background: To determine the extent to which hepatic stellate cell (HSC) activation contributes to liver fibrosis, it was found necessary to develop an alternative structural and functional stellate cell marker for in situ studies. Although several HSC markers have been reported, none of those are associated with particular HSC functions.