Reduction of connexin 43 expression in aged human dental pulp

AIM: To investigate the expression of connexin 43 (CX43) mRNA in young and old human dental pulp tissues to determine the characteristics of CX43 expression. METHODOLOGY: Samples were obtained from human dental pulp of healthy young (17-23 years) and aged (>50 years) subjects. CX43 expression was determined by RT-PCR and by quantitative real-time RT-PCR (QRT-PCR). The threshold cycle (Ct) value, which reflects the amount of PCR, was calculated and the difference between value in the young pulp and that in the aged pulp was statistically analysed. RESULTS: RT-PCR analysis of human dental pulp tissue detected CX43 mRNA in all the samples. CX43 was abundantly expressed in young adult dental pulp, but expression of CX43 mRNA was dramatically decreased in aged human dental pulp. QRT-PCR analysis also showed the reduced expression of CX43 in aged pulp, and expression of CX43 in young pulp was significantly higher (about 10-fold, P < 0.01, Mann-Whitney U-test). CONCLUSION: Reduction of CX43 expression may be associated with the loss of viability in human dental pulp, and is considered as one characteristic of aged pulp.     

Hypoxia induces expression and activation of AMPK in rat dental pulp cells

AMP-activated protein kinase (AMPK) is a stress-responsive enzyme involved in cell adaptation to an energy crisis. We hypothesized that hypoxia suppresses oxidative phosphorylation and ATP production, resulting in AMPK activation to protect cells. We investigated the effects of hypoxia on cell proliferation, the expression of AMPK and hypoxia-inducible factor 1alpha (HIF-1alpha), the activation of AMPK, and the relationship between AMPK and HIF-1alpha expression in rat dental pulp RPC-C2A cells. AMPK in the cells was composed of catalytic alpha1, and regulatory beta1 and gamma1 subunit isoforms. Cell proliferation was initially suppressed under hypoxia, but it increased thereafter, together with an increase in the expression of AMPK and HIF-1alpha, and the activation of AMPK. Down-regulation of AMPKalpha1 by siRNA inhibited cell proliferation under both normoxia and hypoxia, revealing that AMPK induction and activation were required for cell proliferation, although HIF-1alpha expression under hypoxia was not affected.     

Morphologic study of cytoskeletal systems of mouse hepatocytes using polyethylene glycol-embedding method

SM mouse livers extracted by immersion in 1% Triton X-100, or in 1% Triton X-100 followed by 0.3 M KI were studied electron microscopically using the polyethylene glycol-embedding method. After extraction with 1% Triton X-100, almost all the structural components of hepatocytes remained intact and cytoplasmic filaments could be seen three-dimensionally by using stereopairs of micrographs. It was difficult, however, to discriminate microfilaments, intermediate-sized filaments and microtubules from one anoter in these specimes . By immersion in 1% Triton X-100 followed by 0.3 M KI, hepatocytes were extracted remaining only plasma membranes, vesicles and filaments. These filaments were approximately 10 nm in diameter, that is intermediate in size. They were branched and were connected with plasma membranes, especilly at desmosomes. The combination method of immersion extraction and PEG-embedding seems to be suitable for the electron microscopic observation of the cytoskeleton of cells in situ.     

Preoperative assessment of hemifacial spasm by the coronal heavily T2-weighted MR cisternography

Background

Microvascular decompression (MVD) has become a well-established surgical procedure for hemifacial spasm (HFS). Before surgery, it is essential to evaluate any possible deformity of the brainstem and establish the precise location of the offending vessels. In the present study of HFS patients we examined coronal sections taken by heavily T2-weighted MR cisternography in addition to routine axial sections, and assessed the usefulness of these images through comparison with intraoperative findings.

Methods

Eighty patients with HFS underwent preoperative coronal heavily T2-weighted MR cisternography before microvascular decompression surgery. Three neurosurgeons examined the preoperative axial and coronal MR images and evaluated vessel invagination into the brainstem. The usefulness of coronal sections was assessed statistically by the Mann-Whitney U test.

Results

Invagination of the offending vessel into the brainstem was observed in 24 cases (30.0%). In 19 patients, it was predicted preoperatively that compression of the flocculus and brainstem would be required in order to approach the offending vessels. Coronal MR cisternography was significantly more useful in cases with vessel invagination into the brainstem than in cases without invagination.

Conclusions

Coronal sections obtained by MR cisternography are able to demonstrate the severity of vessel invagination into the brainstem as well as revealing the presence of the offending vessel. This information is helpful for planning a suitable approach to the root exit zone.     

Enhanced tumor cell selectivity of adriamycin-monoclonal antibody conjugate via a poly(ethylene glycol)-based cleavable linker.

A novel linker consisting of poly(ethylene glycol) (PEG) and dipeptide was used for conjugation of adriamycin with tumor-specific monoclonal antibody, NL-1, to confirm that the linker can be cleaved selectively with the tumor specific enzyme to express cytotoxicity of the anti-tumor agent. Initially, adriamycin-conjugated PEG linkers through different amino acid compositions, alanyl-valine (Ala-Val), alanyl-proline (Ala-Pro), and glycyl-proline (Gly-Pro) sequences, were prepared to confirm selective digestion with model enzymes. Adriamycin was released by particular model endoproteases, thermolysin and proline endopeptidase, from the linkers with different efficiency. When conjugates were prepared using these adriamycin-bound linkers, conjugates had no loss of binding affinity and specificity for common acute lymphoblastic leukemia antigen (CALLA) expressed on the Daudi cell surfaces as the target of NL-1 antibody. In addition, adriamycin release from the conjugates was also confirmed by incubating them with specific proteases. Tumor cell growth was inhibited dose-dependently for the conjugates carrying Ala-Val and Gly-Pro linkers, whereas significant inhibitory effect was abolished for the conjugate carrying Ala-Pro linker, indicating that cytotoxic effect can be controlled by specificity of antibody and composition of linker peptide. IC(50) for Ala-Val linked conjugate was approximately 3.5 microg/ml and that for Gly-Pro linked conjugate was 5.2 microg/ml. PEG-dipeptidyl linker demonstrated here will be an effective tool for the preparation of immunoconjugate, especially specific activation of anti-tumor agents at desired tumor tissues.     

Lysosomal membrane permeabilization causes secretion of IL-1β in human vascular smooth muscle cells

Objective

IL-1β secretion by the inflammasome is strictly controlled and requires two sequential signals: a priming signal and an activating signal. Lysosomal membrane permeabilization (LMP) plays a critical role in the regulation of NLRP3 inflammasome, and generally acts as an activating signal. However, the role of LMP controlling NLRP3 inflammasome activation in human vascular smooth muscle cells (hVSMCs) is not well defined.

Methods

LMP was induced in hVSMCs by Leu-Leu-O-methyl ester. Cathepsin B was inhibited by CA-074 Me. Cytokine release, mRNA, and protein were quantified by enzyme-linked immunosorbent assay, quantitative PCR, and Western blot, respectively. NF-κB activity was analyzed by immunostaining of the NF-κB p65 nuclear translocation and using the dual-luciferase reporter assay system.

Results

LMP had both priming and activating roles, causing an upregulation of proIL-1β and NLRP3 and the secretion of mature IL-1β from unprimed hVSMCs. LMP activated the canonical NF-κB pathway. The priming effect of LMP was inhibited by CA-074 Me, indicating an upstream role of cathepsin B.

Conclusions

These data support a novel role of LMP as a single stimulus for the secretion of IL-1β from hVSMCs, implying the possibility that hVSMCs are an important initiator of the sterile inflammatory response caused by lysosomal disintegration.

     

Inhibition of vascular adhesion protein-1 enhances the anti-tumor effects of immune checkpoint inhibitors

Modulation of the immunosuppressive tumor microenvironment (TME) is essential for enhancing the anti-tumor effects of immune checkpoint inhibitors (ICIs). Adhesion molecules and enzymes such as vascular adhesion protein-1 (VAP-1), which are expressed in some cancers and tumor vascular endothelial cells, may be involved in the generation of an immunosuppressive TME. In this study, the role of VAP-1 in TME was investigated in 2 murine colon cancer models and human cancer cells. Intraperitoneal administration of the VAP-1-specific inhibitor U-V296 inhibited murine tumor growth by enhancing IFN-γ-producing tumor antigen-specific CD8⁺ T cells. U-V296 exhibited significant synergistic anti-tumor effects with ICIs. In the TME of mice treated with U-V296, the expression of genes associated with M2-like macrophages, Th2 cells (Il4, Retnla, and Irf4), angiogenesis (Pecam1), and fibrosis (Acta2, Loxl2) were significantly decreased, and the Th1/Th2 balance was increased. H₂O₂, an enzymatic product of VAP-1, which promoted the production of IL-4 by mouse Th2 and inhibited IFN-γ by mouse Th1 and human tumor-infiltrating lymphocytes, was decreased in tumors and CD31⁺ tumor vascular endothelial cells in the TMEs of mice treated with VAP-1 inhibitor. TCGA database analysis showed that VAP-1 expression was a negative prognostic factor in human cancers, exhibiting a significant positive correlation with IL-4, IL4R, and IL-13 expression and a negative correlation with IFN-γ expression. These results indicated that VAP-1 is involved in the immunosuppressive TMEs through H₂O₂-associated Th2/M2 conditions and may be an attractive target for the development of combination cancer immunotherapy with ICIs.