Shogo Katsuda

Fas-Mediated Apoptosis in Adriamycin-Induced Cardiomyopathy in Rats In Vivo Study

BACKGROUND The precise molecular mechanism of Adriamycin-induced cardiomyopathy (ADR-CM) is still unknown. We address the demonstration of apoptotic myocardial cell death and the apoptosis-inducing molecules in ADR-CM induced in rats. METHODS AND RESULTS Until 8 weeks after the first administration of ADR, there was no increase in the number of labeled cells by terminal deoxynucleotidyl transferase assay (TUNEL method). Apoptotic indices increased significantly at weeks 9 and 10 in hearts of the ADR-treated group but not in those of the control group (0.42+/-0.12% versus 0.10+/-0.02% and 0.86+/-0.11% versus 0.09+/-0.04% at weeks 9 and 10, respectively). DNA ladder formation was also observed in the myocardial tissues during the late stages of the ADR-CM of rats. There was no significant difference in expression of p53 gene between the ADR group and the control group at either the message or the protein level. An overexpression of Fas antigen was shown in myocardial cells of ADR-treated hearts at weeks 9 and 10 by both Western blotting and immunofluorescent staining. Furthermore, we confirmed that neutralization of anti-Fas ligand antibody inhibited ADR-induced apoptosis. CONCLUSIONS Apoptotic cell death was observed in the hearts of ADR-CM rats, and the number of apoptotic myocardial cells increased with the deterioration of morphological findings and cardiac function, indicating that apoptosis may be an important mechanism of loss of myocardial cells and cardiac dysfunction in ADR-CM. Apoptosis in ADR-CM rats is not p53-dependent but rather is executed through a Fas-mediated pathway.

FDG PET in the evaluation of the aggressiveness of pulmonary adenocarcinoma : correlation with histopathological features

2-[Fluorine-18]fluoro-2-deoxy-d-glucose (FDG) uptake within the primary lesion correlates with survival on positron emission tomography (PET) studies of patients with non-small cell lung cancer. The more metabolically active the tumour, the worse the outcome. The aim of this study was to determine whether a correlation exists between aggressiveness as determined by pathology and the findings of FDG PET in pulmonary adenocarcinoma. Thirty-five patients with 38 adenocarcinomas of the lung were studied. All patients underwent thoracotomy within 4 weeks of the FDG PET study. For semiquantitative analysis, standardized uptake values (SUVs) were calculated. Patients were classified into high SUV (⩾4.0) and low SUV (<4.0) groups. The degree of FDG uptake (SUVs) in primary lung lesions was correlated with the histopathological features of aggressiveness (pleural involvement, vascular invasion or lymphatic permeation). The mean SUV of aggressive adenocarcinomas (4.36±1.94, n = 22) was higher than that of non-aggressive ones (1.53±0.88, n = 16) (P<0.0001). Tumours with a high FDG uptake have a significantly higher likelihood of aggressiveness than those with a low FDG uptake (P = 0.0004). Analysis by the Kaplan-Meier methods revealed that the groups had different prognoses (log-rank test, P = 0.0099). The high SUV group had a significantly worse prognosis. In conclusion, a correlation was seen between aggressiveness as determined by pathology and glucose metabolism as measured by FDG PET in adenocarcinoma of the lung. FDG PET may be used as a non-invasive diagnostic technique in measuring aggressiveness and prognosis in patients with pulmonary adenocarcinoma.

Expression of glial fibrillary acidic protein gfap in peripheral nerve sheath tumors a comparative study of immunoreactivity of gfap vimentin s 100 protein and neurofilament in 38 schwannomas and 18 neurofibromas

Immunoreactivity of glial fibrillary acidic protein (GFAP) in 38 schwannomas and 18 neurofibromas was evaluated and compared with the reactivity of vimentin, S-100 protein, and neurofilament protein. All cases were positive for vimentin and S-100 protein. GFAP was positively stained in the neoplastic cells of 15 of 38 schwannomas (38%) and in two of 18 neurofibromas (11%). The extensively stained GFAP-positive tumors tended to be deeply situated in the body. The GFAP-positive cells were usually spindle-shaped and appeared preferentially in the perivascular region of hyalinized, thick blood vessels.

Relationship of aquaporin 1, 3, and 5 expression in lung cancer cells to cellular differentiation, invasive growth, and metastasis potential

An oncogenic capacity of aquaporins, transmembrane channels for water, was recently proposed. This study seeks to elucidate the involvement of aquaporin 1, 3, and 5 in the development and progression of lung cancer. Expression of aquaporin 1, 3, and 5 was examined by immunohistochemistry, Western blot, and laser-captured microdissection/real-time reverse transcription polymerase chain reaction in 160 lung cancers of various histologic subtypes; and its correlation with clinicopathological factors and survival was analyzed. Aquaporin 1, 3, and 5 were expressed in tumor cells in 71%, 40%, and 56% of lung cancers, respectively. Aquaporin expressions were frequent in adenocarcinomas, whereas aquaporin 1 and 5 were negative in squamous cell carcinomas. Bronchioloalveolar carcinoma cells exhibited an apicolateral aquaporin 1 and apicolateral or basolateral aquaporin 3 localization in nonmucinous type, and apical aquaporin 1 and 5 and basolateral aquaporin 3 expression in mucinous type, which corresponded to aquaporins expression of nonneoplastic lung tissue. Basolateral aquaporin 5 expression was acquired during tumorigenesis of nonmucinous bronchioloalveolar carcinoma. In contrast, invasive adenocarcinoma tumor cells overexpressed aquaporin 1 and 5 with loss of subcellular polarization and with an intracytoplasmic distribution. Overexpression of aquaporin 1 correlated with high postoperative adenocarcinoma metastasis ratios and unfavorable disease-free survival rates (P = .031). We conclude that expression patterns of aquaporin 1, 3, and 5 in lung cancer cells are mostly associated with cellular differentiation. However, the expression of aquaporin 1 and 5 is up-regulated in invading lung cancer cells, particularly in adenocarcinomas; and the overexpression of aquaporin 1 with loss of subcellular polarization is suggested to be involved in their invasive and metastatic potential.

Active immunization of combined β1-adrenoceptor and M2-muscarinic receptor peptides induces cardiac hypertrophy in rabbits

BACKGROUND The high prevalence of patients with dilated cardiomyopathy (DCM) with anti-beta1-adrenoceptor and/or anti-M2-muscarinic receptor autoantibodies in their sera has been observed. However, the pathophysiological role of these autoantibodies in the development of cardiomyopathy is unknown. We previously reported an experimental model of early-stage DCM-like cardiomyopathy induced by immunizing rabbits for 1 year with synthetic peptides corresponding to the sequence of the second extracellular loop of either beta1-adrenoceptor or M2-muscarinic receptor. Because approximately half the sera of patients with DCM that recognize one of the two receptor sequences also recognize the second sequence, a model was created in rabbits simultaneously immunized with the synthetic peptides corresponding to the second extracellular loop of the beta1-adrenoceptor and M2-muscarinic receptor. METHODS AND RESULTS All rabbits (n = 8) immunized with both peptides had a high titer of both anti-beta1-adrenoceptor and anti-M2-muscarinic receptor autoantibodies in their sera, whereas none of the sera from control rabbits injected with saline (n = 9) was positive. No significant cross-reaction with peptides other than those used for immunization was found. The weight of the hearts of immunized rabbits increased significantly. The hearts of immunized rabbits showed marked concentric left ventricular hypertrophy with mild inflammatory cell infiltration. In these rabbits, mild or moderate interstitial fibrosis was also observed. In electron micrographs, immunized rabbits showed focal myofibrillar lysis, loss of myofilament, and a marked increase in the number of mitochondria and deposition of dense granules in both sarcoplasm and myofibrils. Conversely, one of the control rabbits showed scant mononuclear cell infiltration. However, in this control rabbit, no significant alteration was found by electron microscopy. CONCLUSION Our results showed the coexistence of both anti-beta1-adrenoceptor and anti-M2-muscarinic receptor autoantibodies in the sera has pathophysiological importance, shown by their ability to induce cardiac hypertrophy in rabbits.

Expression of ADAMTS-4 (aggrecanase-1) and possible involvement in regression of lumbar disc herniation.

Study Design. Examination of ADAMTS-4 expression, and cellular lineages, distribution, and numbers of ADAMTS-4 (aggrecanase-1) expressing cells in herniated lumbar intervertebral discs. Objective. To determine the expression of ADAMTS-4, a metalloproteinase capable of digesting aggrecan, and its role in herniated lumbar intervertebral disc degradation. Summary of Background Data. Matrix metalloproteinases degrade extracellular matrix of herniated discs, but the mechanism of aggrecan degradation, the major component of intervertebral discs, is poorly understood. Methods. Surgically resected herniated lumbar intervertebral discs from 22 patients were subclassified into protrusion, subligamentous extrusion, transligamentous extrusion, and sequestration types. Reverse transcriptase polymerase chain reaction, Western blot, and immunohistochemistry were used to evaluate ADAMTS-4 messenger ribonucleic acid and protein expression. Results. Expression of ADAMTS-4 messenger ribonucleic acid and protein was shown in the samples of herniated lumbar intervertebral discs. Immunohistochemical staining showed that ADAMTS-4 was mainly localized in CD68-positive mononuclear cells in granulation and adjacent disc tissues. ADAMTS-4 positive cell counts were significantly higher in transligamentous extrusion and sequestration than protrusion and subligamentous extrusion types. Alcian blue staining showed a decrease of proteoglycan in transligamentous extrusion and sequestration cases. Conclusions. Macrophages infiltrating granulation and adjacent disc tissues express ADAMTS-4, suggesting its involvement in herniated disc regression.

Collagen Synthesis by Cultured Arterial Smooth Muscle Cells during Spontaneous Phenotypic Modulation

Quantitative and qualitative changes incollagen synthetic activity by rabbit arterial smooth muscle cells were monitored during spontaneous phenotypic modulation from days 2‐15 of culture. The cultured smooth muscle cells transformed into a synthetic phenotype, reaching a maximum of 94.6% on day 4, and then gradually returned to a conractile phenotype accounting for 59.3% on day 15 of culture. The maximum collagen synthesis was found on day 7 when the cells were in early quiescent phase and showed a 91.7% synthetic phenotype. With an increasing proportion of cells decreased in parallel with the reduction in total protein synthesis. Synthesis of type I collagen was predominant, and the proportion of type I+III collagen was over 85% during the entire period of culture. Synthetic activity of type IV collagen, however, was relatively increased, and reached 3.8±0.4% a t day 15 in comparison with 0.81±0.1% in the late logarithmic growth phase on day 4. This significant increment of type IV collagen in vitro seems to be correlated with the phenotypic modulation of cultured smooth muscle cells into a contractile phenotype. Acta Pathol Jpn 40: 157‐164, 1990.

Collagen synthesis of human arterial smooth muscle cells: Effects of platelet-derived growth factor, transforming growth factor-β1 and interleukin-1

The effects of platelet‐derived growth factor (PDGF), transforming growth factor‐β1 (TGF‐β1) and interleukin‐1 (IL‐1) on collagen synthesis of cultured human arterial smooth muscle cells in a confluent state were investigated. Synthetic activity of collagenous protein was determined with [3H]‐proline uptake, and subsequent analysis of collagen types by sodium dodecylsulfte‐polyacrylmide gel electrophoresis (SDS‐PAGE) followed by fluorography. Although PDGF (0.5 U/mL and 5.0 U/mL) enhanced total collagen synthesis per dish, it suppressed total collagen synthesis per DNA (DNA content in a dish). TGF‐β1 (10 pmol/L and 100 pmol/L) enhanced total collagen synthesis both per dish and per DNA. IL‐1 (0.1 U/mL and 1.0 U/mL) suppressed total collagen synthesis both per dish and per DNA. A fluorogram revealed that human arterial smooth muscle cells synthesize types I, III, IV and V collagen. Densitometric analysis showed PDGF suppressed the proportion of type V collagen. TGF‐β1 increased the proportions of types IV and V collagen. IL‐1 elicited un‐remarkable change in the proportion of collagen types. These results suggest that, in the event of human atherosclerosis, TGS‐β1 is most effective in enhancing collagen synthesis, and PDGF modulates collagen metabolism by stimulating a cell division of smooth muscle cells with a resultant increase of collagenous protein, especially of type V collagen.

An elastinolytic enzyme detected in the culture medium of human arterial smooth muscle cells

The culture medium of human arterial smooth muscle cells exhibits an elastinolytic activity with 68 and 64 kDa on elastin substrate gels. The enzymatic activities are inhibited by ethylenediamine tetraacetic acid, a metalloproteinase inhibitor, but not by other inhibitors of serine, cysteine and aspartic proteinases. The proteinase in the culture medium is activatable by 4‐aminophenylmercuric acetate and degrades insoluble elastin. Compared to other matrix metalloproteinases (MMP), the activity shows the similar elastinolytic pattern to that by MMP‐2 purified from human rheumatoid synovium, while MMP‐3 and MMP‐9 have different lytic patterns and MMP‐1 possesses no elastinolytic activity. An immunoblot analysis demonstrated that the 68‐kDa enzyme is MMP‐2. An immunofluorescence study illustrates that MMP‐2 is localized within the cytoplasm of the smooth muscle cells. These findings suggest that the elastinolytic enzyme secreted by human arterial smooth muscle cells is MMP‐2.

Mechanism of gallium 67 accumulation in inflammatory tissue

The present study was undertaken to elucidate the accumulation mechanism of gallium 67 in inflammatory tissue.67Ga accumulation in inflammatory tissue was observed by macro- and microautoradiogram. Permeability indices were calculated for serum albumin from blood vessels into inflammatory and normal tissue. Neutrophils and macrophages did not play a major role in67Ga accumulation in the inflammatory tissue because67Ga could hardly be detected in the sites in which neutrophils were crowded; the accumulation was concentrated in the intercellular space around these cells in the tissue. Permeability indices for inflammatory tissue were much greater than those for normal tissues. It is thought from the present study and previously reported results that67Ga, together with plasma from permeable blood vessels, readily penetrates the inflammatory tissue and stays there by binding to the acid mucopolysaccharide present in the tissue.