Seiji Shimada

The Relationship Between Microvessel Density, the Expression of Vascular Endothelial Growth Factor (VEGF), and the Extension of Nasopharyngeal Carcinoma

Objective The present study was aimed at clarifying whether the microvessel density (MVD) and the e‐pression of vascular endothelial growth factor (VEGF) were related to the degree of local invasion and metastasis in nasopharyngeal carcinoma (NPC).

Argatroban, specific thrombin inhibitor, induced phenotype change of cultured rabbit vascular smooth muscle cells

To investigate whether argatroban ((2R,4R)-4-methyl-1-[N(2)-((RS)-3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfonyl)-L-arginyl]-2-piperidinecarboxylic acid hydrate, a selective thrombin inhibitor, exerts a direct action on phenotype conversion of vascular smooth muscle cells, cultured rabbit aortic vascular smooth muscle cells were employed. Myosin heavy chain isoforms (SM1, SM2, and SMemb) mRNA expressions were evaluated by in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR). After the cells were incubated in serum-free medium containing argatroban (10 and 50 microg/ml) and platelet-derived growth factor (PDGF)-BB (10 and 50 ng/ml) for 3 h, total RNA was extracted. In situ hybridization demonstrated that myosin heavy-chain isoform mRNAs were homogenously expressed in argatroban- and PDGF-BB-treated cells. RT-PCR revealed that SM1/SM2 mRNA expressions were not changed with argatroban, while SMemb mRNA expression was increased to 1.6-fold with a statistical significance (P<0.05). Treatment with argatroban (10 and 50 microg/ml) at 24 h did not change SM1/SM2 mRNA expressions. Although SMemb mRNA expression was slightly increased, there was no statistical significance. Other phenotype markers including plasminogen activator inhibitor-1 (PAI-1) and beta-actin mRNAs were also significantly increased by argatroban. In conclusion, argatroban can directly induce phenotype conversion of vascular smooth muscle cells with the resultant up-regulation of SMemb, PAI-1, and beta-actin mRNAs.

RNA interference targeting embryonic myosin heavy chain isoform inhibited mRNA expressions of phenotype markers in rabbit cultured vascular smooth muscle cells.

To investigate whether the knockdown of SMemb gene expression induces phenotypic modulation of vascular smooth muscle (VSM) cells toward a contractile type, we constructed a siRNA targeting the 3′ untranslated region (UTR) of SMemb gene (SMemb-siRNA). The SMemb-siRNA was introduced into cultured rabbit VSM cells for 48 h at 37°C by the lipofection method. The mRNA expressions were estimated by comparative reverse transcription-polymerase chain reaction (RT-PCR). SMemb-siRNA significantly decreased the ratio of SMemb to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in a dose-dependent manner (P < 0.01): 0 nM, 0.90 ± 0.08; 100 nM, 0.43 ± 0.07. Immunofluorescence and immunoblot analyses demonstrated that SMemb-siRNA markedly decreased SMemb protein expression to 56% ± 7.8% (P < 0.01). Other MHC isoform (SM1 and SM2) mRNA expressions were not changed. The relative mRNA expressions of other phenotype markers (plasminogen activator inhibitor (PAI)-1 and β-actin) were significantly decreased by SMemb-siRNA to 71% ± 7.5% and 61% ± 7.5%, respectively (P < 0.01). Expression of smooth muscle (SM) α-actin protein and cell proliferation was not changed by SMemb-siRNA. Thus, SMemb gene might be involved in the transcription of PAI-1 and β-actin, but not involved in SM α-actin and cell proliferation in cultured VSM.

The Nucleotide Sequence of Dinitrophenyl-Specific IgE and FcεRI α-Subunit Obtained from FE-3 Hybridoma Cells

FE-3 cells were established by Hanashiro et al. by hybridizing mouse myeloma cells (Sp2/0-Ag14/SF) with rat spleen cells that were freshly isolated from Brown–Norway rats sensitized with DNP-As. FE-3 cells can constitutively secrete IgE without stimulation by cytokines. Our preliminary experiments demonstrated that the IgE secretion was decreased at 3 days after start of culture and the addition of exogenous IgE into culture media depressed the secretion of IgE. Thus, we hypothesized that the IgE production in FE-3 cells may be regulated by a signal transduction through the binding of IgE to its high affinity receptor (FceRI) or to an IgE binding protein on the cell surface. In this study, we aimed to identify the nucleotide sequence of IgE FE-3 and compared with those of mouse IgE and IgE IR162 to find a structural heterogeneity in the Fc region of IgE FE-3. We also tested if the mRNA of FceRI was expressed in FE-3 cells using the reverse transcriptase-polymerase chain reaction (RT-PCR) method with the c...

RNAi targeting embryonic myosin heavy chain isoform inhibited bound thrombin-induced migration of vascular smooth muscle cells.

To investigate the effect of bound thrombin, a complex of α-thrombin with fibrin fragments derived from clots, on proliferation and migration of cultured rabbit vascular smooth muscle cells, cell proliferation was measured by WST-1 reagent and migration was evaluated by counting migrated cells through pores of cell culture insert (8 μm size) after 48-hour treatment with bound thrombin (10 U/ml). To examine the role of an embryonic myosin heavy chain isoform (SMemb) in these effects by bound thrombin, the cells were subsequently treated for 48 h with an siRNA expression vector (ORF-2/pSilencer) directed against the open reading frame of SMemb mRNA. SMemb and plasminogen activator inhibitor-1 mRNA expressions were measured by Northern blot analysis. Bound thrombin significantly increased SMemb mRNA expression by 1.4 ± 0.01-fold and significantly increased plasminogen activator inhibitor-1 mRNA expression by 2.65 ± 0.69-fold (p < 0.01 vs. PBS treatment for each), which were abolished by treatment with ORF-2/pSilencer. Although bound thrombin had no effect on cell proliferation, bound thrombin significantly increased migration by 1.93 ± 0.20-fold (p < 0.05). ORF-2/pSilencer treatment significantly reduced the bound thrombin-stimulated migration activity by 1.28 ± 0.15-fold (p < 0.05). Thus, SMemb plays an important role in bound thrombin-induced cell migration activity of cultured vascular smooth muscle cells.