Yuri Akishima

Immunohistochemical detection of human small lymphatic vessels under normal and pathological conditions using the LYVE-1 antibody.

The spread of tumor cells via lymphatic vessels to the lymph nodes is an important indicator of malignancy. However, previous markers used to identify lymphatic endothelium gave ambiguous results in immunohistochemical analyses with paraffin-embedded tissues. In this study, we attempted to prepare a polyclonal antibody against human lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) for detecting lymphatic vessels using immunohistochemistry. The antibody was raised against a region near the transmembrane anchor of LYVE-1 in New Zealand white rabbits. Immunostainings with anti-LYVE-1 and von Willebrand factor antibodies were performed in various normal and pathological tissues. LYVE-1 expression was confined to the endothelial surface of lymphatic vessels but was not found in the endothelium of blood vessels, which were positive for von Willebrand factor. Our LYVE-1 polyclonal antibody was useful for the identification of small lymphatic vessels in normal human tissues. In addition, the immunostaining enabled us to distinguish lymphatic invasion by malignant tumor cells from blood vessel invasion using paraffin-embedded sections. In conclusion, our polyclonal antibody against the transmembrane anchor of the peptide can be used to detect human lymphatic vessels under various conditions.

Changes in the distribution pattern of gelatin-binding protein of 28 kDa (adiponectin) in myocardial remodelling after ischaemic injury.

Aims:  Gelatin‐binding protein of 28 kDa (GBP28) is a collagen‐like plasma protein having a binding capacity with collagens. We investigated GBP28 role on myocardial remodelling as well as the diagnostic significance of GBP28 immunostaining in myocardial infarction.

Detection of apoptosis in keloids and a comparative study on apoptosis between keloids, hypertrophic scars, normal healed flat scars, and dermatofibroma.

Recent studies have suggested that the regulation of apoptosis during wound healing is important in scar establishment and the development of pathological scarring. In this study, we demonstrate that keloid fibroblasts can be identified as apoptotic cells because of their highly condensed chromatin and discrete nuclear fragments. To further reveal the phenomenon of apoptosis, we quantified the number of terminal deoxynucleotide transferase‐mediated dUTP nick‐end labeling (TUNEL)‐positive cells in surgically resected tissues of keloids (N = 10), hypertrophic scars (N = 10), normal healed flat scars (N = 10), and dermatofibroma (N = 10). The number of TUNEL‐positive cells was relatively low, but was significantly higher for the keloid group compared with the normally healed flat scar group (p = 0.004), suggesting reduced cell survival and increased apoptotic cell death in a subpopulation of keloid fibroblasts. Furthermore, the number of TUNEL‐positive cells was significantly higher for the keloid group compared with the dermatofibroma group (p = 0.044), suggesting that a subpopulation of keloid fibroblasts may suppress tumorgenicity at a greater rate than dermatofibroma by undergoing cell death. Hypertrophic scars had significantly higher levels of apoptosis than normally healed flat scars (p = 0.033). Therefore, these results suggest that selected fibroblasts in keloids and hypertrophic scars undergo apoptosis, which may play a role in the process of pathological scarring.

Role of macrophage and smooth muscle cell apoptosis in association with oxidized low-density lipoprotein in the atherosclerotic development

To examine the role of the apoptosis of macrophages and smooth muscle cells in the development of atherosclerosis, human aortic tissues with intimal lesions were immunostained with antibodies against terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL), single-stranded DNA (clone F7-26), and active caspase-3. Apoptotic cells were detected in the intima using both TUNEL and single-stranded DNA, however, the latter method was the more sensitive one for detecting apoptotic cells in the early stages of atherosclerosis. The number of apoptotic cells increased as the disease progressed. It implies that the apoptosis of intimal cells is involved in the formation of atherosclerotic lesions. In addition, quantitative analyses of the cell types undergoing apoptosis using double-immunostaining revealed that the susceptibility of macrophages and smooth muscle cells to apoptosis was greater specifically in atheroma than in the other atherosclerotic lesions, and macrophages were more susceptible to apoptosis than smooth muscle cells. The frequency and spatial distribution of oxidized low-density lipoprotein (oxLDL) (FOH1a/DLH3)-positive cells were examined by immunohistochemistry, and the results resembled those of apoptotic cells. The number of oxLDL-positive cells in the intima significantly correlated with the susceptibility of smooth muscle cells, but not with that of macrophages, to apoptosis. These results suggest that oxLDL affects the apoptosis of smooth muscle cells during the atherosclerotic development.

The distribution and production of cholesteryl ester transfer protein in the human aortic wall

Cholesteryl ester transfer protein (CETP) has been considered to mediate the transfer of cholesteryl ester from arterial wall, however, the distribution and production of CETP in human arterial wall remains unclear. Present study histopathologically demonstrated the distribution of CETP and CETP mRNA in the human aortic wall by immunohistochemistry and in situ hybridization. While CETP was constantly distributed in the media, the protein was recognized within the intima with fibrocellular thickening and atherosclerotic intima. Double immunostaining methods demonstrated CETP expression in smooth muscle cells in the intima and media. CETP mRNA was detected not only in intimal cells but medial smooth muscle cells. Intimal cells expressing CETP mRNA were considered to be monocyte-derived macrophages and smooth muscle cells by immunohistochemistries using two antibodies against smooth muscle actin and human macrophage on the subserial sections. Our in vivo study provides that CETP is produced by smooth muscle cells in the intima and media of human aorta, and it is suggested that arterial smooth muscle cells positively participate in the removal of excessive cholesteryl ester from the arterial wall by CETP production.

Activated caspase expression and apoptosis increase in keloids: cytochrome c release and caspase-9 activation during the apoptosis of keloid fibroblast lines.

To characterize apoptosis in keloids and the mechanisms responsible for this process, the expression of activated caspase‐9 and ‐3 in fibroblasts obtained from keloids was analyzed. Immunohistochemistry revealed that the number of fibroblasts positive for terminal deoxynucleotide transferase‐mediated dUTP nick‐end labeling (TUNEL) or activated caspase‐9 or ‐3 was low but was significantly higher in keloid tissues than in normal scar tissues. Significant relationships between the number of caspase‐positive fibroblasts and TUNEL‐positive fibroblasts suggested that the activation of caspase‐9 and ‐3 induces apoptosis in a subpopulation of keloid fibroblasts. All keloid fibroblast cell lines established in this study showed activation of caspase‐9 and ‐3 after serum deprivation for 3 or 4 hours, as shown using Western blotting. Furthermore, serum deprivation‐induced apoptosis in a keloid fibroblast line was blocked by a caspase‐9 inhibitor (acetyl‐Leu‐Glu‐His‐Asp‐al), indicating that activation of caspase‐9 was necessary for the process of apoptosis in keloid fibroblasts. Although serum deprivation did not significantly change the level of apoptosis protease activating factor‐1 in any of the lines, cytochrome c release was detected in cytosolic fractions of the lines after serum deprivation for 3 or 4 hours. These results strongly suggest that keloid fibroblasts are predisposed to apoptosis and cytochrome c release and that caspase‐9 activation may underlie regulation of apoptosis in keloid fibroblasts in vivo.

Expression of cholesteryl ester transfer protein (CETP) in germinal centre B cells and their neoplastic counterparts

Aims:  Cholesteryl ester transfer protein (CETP) is known to facilitate the transfer of lipids between plasma lipoproteins. Previous studies on human tissues have determined that the spleen contains large amounts of CETP mRNA, while the exact location of CETP in such organs remains unknown. In the present study, our aim was to locate CETP protein expression at the cellular level in human normal and neoplastic lymphoid organs.

A case of pleomorphic adenoma with cystic squamous differentiation.

We report a case of pleomorphic adenoma with cystic squamous differentiation of the parotid gland. Needle aspirates of the tumor showed numerous squamous cells without a mucous background. Cuboidal epithelial cells and stromal cells were few. Histopathologically, cystic change in the pleomorphic adenoma formed a large central space lined with stratified differentiated squamous epithelia. Incorrect diagnosis is prevented by paying sufficient attention to determining benign squamous differentiation in pleomorphic adenoma.