Sadayuki Hiroi

Formation of N ε-(Hexanonyl)lysine in Protein Exposed to Lipid Hydroperoxide A PLAUSIBLE MARKER FOR LIPID HYDROPEROXIDE-DERIVED PROTEIN MODIFICATION

The objectives of this study were to estimate the structure of the lipid hydroperoxide-modified lysine residue and to prove the presence of the adducts in vivo. The reaction of lipid hydroperoxide toward the lysine moiety was investigated employingN-benzoyl-glycyl-l-lysine (Bz-Gly-Lys) as a model compound of Lys residues in protein and 13-hydroperoxyoctadecadienoic acid (13-HPODE) as a model of the lipid hydroperoxides. One of the products, compound X, was isolated from the reaction mixture of 13-HPODE and Bz-Gly-Lys and was then identified as N-benzoyl-glycyl-N ε-(hexanonyl)lysine. To prove the formation of N ε-(hexanonyl)lysine, named HEL, in protein exposed to the lipid hydroperoxide, the antibody to the synthetic hexanonyl protein was prepared and then characterized in detail. Using the anti-HEL antibody, the presence of HEL in the lipid hydroperoxide-modified proteins and oxidized LDL was confirmed. Furthermore, the positive staining by anti-HEL antibody was observed in human atherosclerotic lesions using an immunohistochemical technique. The amide-type adduct may be a useful marker for the lipid hydroperoxide-derived modification of biomolecules.

Characterization of two F4/80-positive Kupffer cell subsets by their function and phenotype in mice

BACKGROUND & AIMS Liver Kupffer cells have been suggested to be heterogeneous macrophage lineage cells. We explored this possibility by classifying the mouse Kupffer cells into subpopulations and characterizing them by their phenotype and function. METHODS Liver mononuclear cells (MNCs) from C57BL/6 mice were isolated and their phenotypes and functions were analyzed. The effects of clodronate liposomes and gadolinium chloride (GdCl(3)) on Kupffer cells were also investigated. RESULTS Approximately 25% of liver MNCs were F4/80(+) Kupffer cells. Of these, 46% were CD11b(-)CD68(+), 22% were CD11b(+)CD68(-), and 6% were CD11b(+)CD68(+). CD68(+) cells showed potent phagocytic activity and reactive oxygen species (ROS) production capacity after lipopolysaccharide (LPS) stimulation, whereas CD11b(+) cells did not. CD11b(+) cells showed a strong capacity for the production of cytokines (TNF and IL-12), which was much less prominent in CD68(+) cells. At 24h after LPS or Escherichia coli injection into mice, the proportions of CD11b(+)CD68(-) and CD11b(+)CD68(+) cells increased but that of CD11b(-)CD68(+) cells decreased. The increase in CD11b(+)CD68(+) cells appeared to be derived from the CD11b(+)CD68(-) subset. Although the CD11b(+) cells augmented phagocytic activity after LPS injection, they did not increase ROS production, suggesting their weak lytic activity. Injection of clodronate or GdCl(3) into mice depleted the CD68(+) cells but increased CD11b(+) cells proportionally because CD68(+) cells may phagocytose these toxic reagents and undergo apoptosis. GdCl(3)-treated mice also consistently increased serum TNF after LPS challenge. CONCLUSIONS Two F4/80(+) Kupffer cell subsets may exist, a CD68(+) subset with phagocytic activity and a CD11b(+) subset with cytokine-producing capacity.

A High-Cholesterol Diet Exacerbates Liver Fibrosis in Mice via Accumulation of Free Cholesterol in Hepatic Stellate Cells

BACKGROUND & AIMS Some studies have indicated that dietary cholesterol has a role in the progression of liver fibrosis. We investigated the mechanisms by which dietary cholesterol might contribute to hepatic fibrogenesis. METHODS C57BL/6 mice were fed a high-cholesterol diet or a control diet for 4 weeks; liver fibrosis then was induced by bile-duct ligation or carbon tetrachloride administration. Hepatic stellate cells (HSCs) were isolated from mice fed high-cholesterol diets or from Niemann-Pick type C1-deficient mice, which accumulate intracellular free cholesterol. RESULTS After bile-duct ligation or carbon tetrachloride administration, mice fed high-cholesterol diets had significant increases in liver fibrosis and activation of HSCs compared with mice fed control diets. There were no significant differences in the degree of hepatocellular injury or liver inflammation, including hepatocyte apoptosis or Kupffer cell activation, between mice fed high-cholesterol or control diets. Levels of free cholesterol were much higher in HSCs from mice fed high-cholesterol diets than those fed control diets. In cultured HSCs, accumulation of free cholesterol in HSCs increased levels of Toll-like receptor 4 (TLR4), leading to down-regulation of bone morphogenetic protein and activin membrane-bound inhibitor (a pseudoreceptor for transforming growth factor [TGF]β); the HSCs became sensitized to TGFβ-induced activation. Liver fibrosis was not aggravated by the high-cholesterol diet in C3H/HeJ mice, which express a mutant form of TLR4; HSCs that express mutant TLR4 were not activated by accumulation of free cholesterol. CONCLUSIONS Dietary cholesterol aggravates liver fibrosis because free cholesterol accumulates in HSCs, leading to increased TLR4 signaling, down-regulation of bone morphogenetic protein and activin membrane-bound inhibitor, and sensitization of HSC to TGFβ. This pathway might be targeted by antifibrotic therapies.

Acceleration of wound healing in healing-impaired db/db mice with a photocrosslinkable chitosan hydrogel containing fibroblast growth factor-2.

Application of ultraviolet light irradiation to a photocrosslinkable chitosan (Az‐CH‐LA) aqueous solution including fibroblast growth factor‐2 (FGF‐2) results within 30 seconds in an insoluble, flexible hydrogel. The FGF‐2 molecules retained in the chitosan hydrogel remain biologically active and are released from the chitosan hydrogel upon in vivo biodegradation of the hydrogel. To evaluate the accelerating effect on wound healing of this hydrogel, full‐thickness skin incisions were made in the backs of healing‐impaired diabetic (db/db) mice and their normal (db/+) littermates. The mice were later killed, and histological sections of the wound were prepared. The degree of wound healing was evaluated using several histological parameters such as the rate of contraction, epithelialization, and tissue filling. Application of the chitosan hydrogel significantly advanced the rate of contraction on Days 0 to 2 in db/db and db/+ mice. Although the addition of FGF‐2 into the chitosan hydrogel in db/+ mice had little effect, application of the chitosan hydrogel–containing FGF‐2 further accelerated the adjusted tissue filling rate (Days 2 to 4 and Days 4 to 8) in db/db mice. Furthermore, the chitosan hydrogel–containing FGF‐2 markedly increased the number of CD‐34‐positive vessels in the wound areas of db/db mice on Day 4. Thus, the application of chitosan hydrogel–containing FGF‐2 onto a healing‐impaired wound induces significant wound contraction and accelerates wound closure and healing.

Expression of Hypoxia-Inducible Factor-1α Protein Predicts Survival in Patients with Transitional Cell Carcinoma of the Upper Urinary Tract

Hypoxia-inducible factor-1 (HIF-1), identified as one of the transcription factors, has been found to play an essential role in oxygen homeostasis. HIF-1 is a heterodimer composed of HIF-1α and HIF-1β. Increased levels of HIF-1α have been reported during the carcinogenesis and progress of several tumors. We investigated the prognostic importance of HIF-1α expression in transitional cell carcinoma of the upper urinary tract. In 127 cases of transitional cell carcinoma of the upper urinary tract, we examined its expression (using immunohistochemistry and in situ hybridization), and also its relation to the expression of p53 oncoprotein, as well as to proliferating cell nuclear antigen (PCNA) immunoreactivity, microvessel density, clinicopathologic parameters, and clinical outcome. A positive expression of HIF-1α protein was recognized in 55.1% of samples, the expression being apparent within the nucleus in tumor cells. HIF-1α protein expression correlated with grade, growth pattern, p53 oncoprotein expression, and PCNA index, but not with stage. Furthermore, a significant correlation was found between HIF-1α protein expression and both overall and disease-free survival rates in the univariate and multivariate analyses (in all tumors and in invasive tumors). A positive expression of HIF-1α mRNA was recognized in 69.6% of 125 samples which were available, the expression being apparent within the cytoplasm in tumor cells. The positive expression of HIF-1α mRNA by in situ hybridization correlated significantly with HIF-1α protein expression by immunohistochemistry. HIF-1α mRNA expression only correlated with pattern of growth (P = 0.0078). In conclusion, the detection of HIF-1α protein would seem to be of value in informing the prognosis of transitional cell carcinoma of the upper urinary tract.

Small interfering RNA targeting epidermal growth factor receptor enhances chemosensitivity to cisplatin, 5-fluorouracil and docetaxel in head and neck squamous cell carcinoma.

Overexpression of epidermal growth factor receptor (EGFR) has been found in various epithelial malignancies, including head and neck squamous cell carcinoma (HNSCC), and is associated with increased tumor growth, metastasis, resistance to chemotherapeutic agents and poor prognosis. As such, EGFR is a potential target for antitumor therapy and several EGFR inhibitors have been investigated in preclinical or clinical settings. In the present study, we used small interfering RNA (siRNA) to downregulate EGFR expression while evaluating the effect of EGFR siRNA on cell proliferation, and the combined effects with cisplatin, 5‐fluorouracil (5‐FU) and docetaxel in HNSCC. Furthermore, HNSCC xenografts were treated with EGFR siRNA alone or in combination with cisplatin, and tumor growth was examined. EGFR expression, proliferation, angiogenesis and apoptosis index were evaluated by immunohistochemistry. The results showed that EGFR siRNA efficiently downregulated EGFR expression and inhibited cell growth of HNSCC. Treatment with EGFR siRNA in combination with cisplatin, 5‐FU and docetaxel enhanced chemosensitivity with a significant increase in apoptosis. EGFR siRNA delivered by atelocollagen enhanced the antitumor effect of cisplatin in the HNSCC xenograft model. These cumulative results suggest that EGFR siRNA combined with cisplatin, 5‐FU and docetaxel may be a feasible strategy to enhance the effects of chemotherapy in patients with HNSCC. (Cancer Sci 2006; 97: 1115–1124)

Expression of LAT1 predicts risk of progression of transitional cell carcinoma of the upper urinary tract

L-type amino acid transporter 1 (LAT1), a neutral amino acid transporter, requires covalent association with the heavy chain of 4F2 cell surface antigen (4F2hc) for its functional form. We investigated the importance of LAT1 and 4F2hc expressions to progression in upper urinary tract cancer. We examined their expressions and their relationships to clinicopathologic parameters and clinical outcome in 124 cases. Positive expressions of LAT1 (protein and messenger ribonucleic acid) and 4F2hc (protein) were recognized in 79.8, 89.5, and 87.9% of tumor samples, respectively. In tumor cells, LAT1 protein was detected either as nodular granules within the cytoplasm or diffusely within the cytoplasm and/or on plasma membrane. In the normal urothelium, its expression was detected as nodular granules within the cytoplasm. A correlation with stage was shown for LAT1 protein expression and for a cooperative expression of LAT1 protein with 4F2hc protein (active form of LAT1 protein). Further, in all tumors, a cooperative expression of LAT1 protein and 4F2hc protein was significantly correlated with both overall and disease-free survival rates in the univariate analysis but not in the multivariate analysis. In conclusion, the detection of the active form of LAT1 protein would appear to be of value in informing the risk of progression in transitional cell carcinoma of the upper urinary tract.

Expression of survivin does not predict survival in patients with transitional cell carcinoma of the upper urinary tract

Abstract. The members of the IAP (inhibitors of apoptosis) family, which includes survivin, have recently emerged as modulators of an evolutionarily conserved step in apoptosis. Survivin is present during embryonic and fetal development, but it is downregulated in normal adult tissues. However, it becomes re-expressed in a variety of cancers. We investigated the prognostic importance of the expression of survivin in transitional cell carcinoma of the upper urinary tract (TCC-UUT). In 126 cases of TCC-UUT, we examined its expression (using immunohistochemistry), and also its relationship with the expressions of bcl-2 oncoprotein, p53 oncoprotein, and proliferating cell nuclear antigen (PCNA) immunoreactivity, clinicopathologic parameters, and clinical outcome. A positive expression of survivin was recognized in 12.7% of samples, a granular pattern being apparent within the cytoplasm of tumor cells. Survivin expression did not correlate with clinicopathologic findings, bcl-2 oncoprotein expression, p53 oncoprotein expression, PCNA index, or prognosis. In the normal urothelium, its expression was not detected. In conclusion, the expression of survivin does not predict prognosis in TCC-UUT.

Expression of membrane-type-1-matrix metalloproteinase and metalloproteinase-2 in nonsmall cell lung carcinomas

For the metastasis and invasion of cancer cells, destruction of extracellular matrix is essential. In this process, collagen is broken down by some matrix metalloproteinases. Matrix metalloproteinase 2 (MMP2) is able to cleave type IV collagen, and membrane-type-1-matrix metalloproteinase (MT1-MMP) induces activation of proMMP2. We investigated the expressions of MT1-MMP and MMP2 and their relation to both clinicopathologic parameters and clinical outcome in non-small cell lung carcinomas (NSCLC). Eighty-nine specimens of NSCLC were examined using in situ hybridization and immunohistochemistry. Each metalloproteinase was expressed within the cytoplasm of tumor cells with or without stromal cells in NSCLC. Tumors in which tumor cells strongly stained for MT1-MMP mRNA or protein made up more than 50% of the tumor area were found in 44 and 26% of cases, respectively. The corresponding values for MMP-2 mRNA and protein, were 51 and 26%. Our analysis of clinicopathological findings revealed a significant positive relationship between MT1-MMP mRNA and p-M. The correlation between MMP2 protein-staining status and overall survival rate reached significance in the univariate analysis. However, an association was not demonstrated in the multivariate analysis. The detection of MT1-MMP and MMP2 is likely to be of limited value in informing the prognosis in NSCLC.

Pivotal advance: characterization of mouse liver phagocytic B cells in innate immunity.

Although B cells in vertebrates have been thought to lack phagocytic activity, there has been a recent report of such ability by the B cells of early vertebrates such as fish and frogs. Here, we show for the first time that mouse liver IgM+ B cells actively phagocytose microsphere beads and Escherichia coli and that they effectively kill bacterial cells. Such phagocytic activity is not observed in other liver MNCs, except for F4/80+ Kupffer cells. In the presence of fresh mouse serum (but not heat‐inactivated serum), the heat‐killed E. coli phagocytic activity of liver B cells increased significantly but was inhibited significantly by anticomplement component C3 antibody, suggesting E. coli opsonization by serum factors, including complement components. Upon i.v. injection of FITC‐labeled E. coli into mice, a substantial proportion of liver B cells phagocytosed the bacteria, as compared with spleen B cells. Functional phagolysosome formation in liver B cells was supported by several reagents showing an acidic change and lysosomes in the phagocytosed vacuoles. Indeed, mouse liver B cells killed viable E. coli more efficiently than did spleen B cells in vitro. Further, E. coli‐phagocytic liver B cells produced a substantial amount of IL‐12. These results indicate that liver B cells have phagocytic and bactericidal activities similar to those of dedicated phagocytes and may contribute to bacterial clearance.