Biomedical subjects

Eiji Warabi

Publications and source records attributed to Eiji Warabi.

7 recordsLinked to original sources

PubMed2026

Inhibition of MAFB and PI3K/AKT Signaling for Hereditary FSGS with Multicentric Carpotarsal Osteolysis.

KEY POINTS: Multicentric carpotarsal osteolysis, a rare disorder, causes progressive osteolysis and kidney failure because of v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) gene mutations. A genome-edited mouse model carrying the multicentric carpotarsal osteolysis mutation was used to obtain a deeper understanding of this rare disease. Targeting MAFB/IGF-1/PI3K/AKT signaling may provide new treatments for multicentric carpotarsal osteolysis-related nephropathy. BACKGROUND: Multicentric carpotarsal osteolysis (MCTO) is a rare condition characterized by progressive osteolysis and often kidney failure. It is caused by autosomal dominant mutations in the transcription factor v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B ( MAFB ). METHODS: Given the absence of efficacious therapeutic interventions for MCTO and the obscurity of its pathophysiologic mechanisms, we used mice with the MCTO mutation ( MafbMCTO/MCTO mice) to explore the role of MAFB. RESULTS: MafbMCTO/MCTO mice displayed FSGS, mirroring the manifestations seen in patients with MCTO. These mice showed that the MCTO mutation leads to the accumulation of MAFB protein. Heterozygous MafbMCTO/- mice, generated by crossbreeding to reduce MAFB levels, neither exhibited albuminuria nor showed any histologic abnormalities in the kidney, suggesting that excess MAFB was detrimental. We subsequently conducted RNA-seq on the glomeruli from MafbMCTO/MCTO mice and detected pronounced upregulation of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway through IGF-1. Given that receptor tyrosine kinases activate PI3K/AKT, we treated MafbMCTO/MCTO mice with the inhibitor imatinib. This led to a significant decline in urinary albumin levels compared with the control group. CONCLUSIONS: Our findings demonstrate that the MCTO mutation resulted in MAFB protein accumulation and led to the development of FSGS in mice.

Animals↗

PubMed2006

Shear stress stabilizes NF-E2-related factor 2 and induces antioxidant genes in endothelial cells: role of reactive oxygen/nitrogen species.

We have previously reported that antioxidant response element (ARE)-regulated genes, such as heme oxygenase 1 (HO-1), sequestosome 1 (SQSTM1), and NAD(P)H quinone oxidoreductase 1 (NQO1), are induced in human umbilical vein endothelial cells (HUVEC) upon exposure to laminar shear stress. In the present study, we have confirmed a critical role for NF-E2-related factor 2 (Nrf2) in the induction of gene expression in HUVEC exposed to laminar shear stress. Although the mRNA levels of Nrf2 were unchanged during exposure to shear stress, the protein levels of Nrf2 were markedly increased. Small interfering RNA (SiRNA) against Nrf2 significantly attenuated the expression of Nrf2-regulated genes such as HO-1, SQSTM1, NQO1, glutamate-cysteine ligase modifier subunit (GCLM), and ferritin heavy chain. Nrf2 was rapidly degraded in cells treated with cycloheximide under static conditions, but shear stress decreased the rate of Nrf2 degradation. Incubation with the thiol antioxidant N-acetylcysteine strongly inhibited both the Nrf2 accumulation and the expression of Nrf2-regulated genes such as HO-1, GCLM, and SQSTM1. Nitric oxide (NO) production was increased with the strength of shear stress but neither the inhibitor of endothelial NO synthase (eNOS) nor the siRNA against eNOS affected the expression of Nrf2-regulated genes. A xanthine oxidase inhibitor oxypurinol and the flavoprotein inhibitor diphenyleneiodonium, which inhibits NAD(P)H oxidase and mitochondrial respiratory chain, markedly suppressed the expression of these genes. Moreover, diphenylpyrenlphosphine, a reducing compound of lipid hydroperoxides, also significantly suppressed Nrf2-regulated gene expression. Taken together, these findings suggest that shear stress stabilizes Nrf2 protein via the lipid peroxidation elicited by xanthine oxidase and flavoprotein mediated generation of superoxide, resulting in gene induction by the Nrf2-ARE signaling pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Tissue Prx I in the protection against Fe-NTA and the reduction of nitroxyl radicals.

Peroxiredoxin I (Prx I) is a key cytoplasmic peroxidase that reduces intracellular hydroperoxides in concert with thioredoxin. To study the role of tissue Prx I in protection from oxidative stress, we generated Prx I-/- mice by gene trapping. We then evaluated the acute-phase tissue damage caused by ferric-nitrilotriacetate (Fe-NTA). Increases in serum aspartate aminotransferase and alanine aminotransferase levels were significantly greater in Prx I-/- than wild-type mice, 4 and 12 h after the injection of Fe-NTA. Using real-time EPR imaging, we examined the reduction of the stable paramagnetic nitroxyl radical 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl in vivo, and found that the half-life of this spin probe in the liver and kidney was significantly prolonged in the Prx I-/- mice. These results demonstrate that Prx I-/- mice have less reducing activity and are more susceptible to the damage mediated by reactive oxygen species in vivo than wild-type mice.

Alanine Transaminase↗

PubMed2005

Differential responses of the Nrf2-Keap1 system to laminar and oscillatory shear stresses in endothelial cells.

The Nrf2-Keap1 system coordinately regulates cytoprotective gene expression via the antioxidant responsive element (ARE). The expression of several ARE-regulated genes was found to be up-regulated in endothelial cells by laminar shear stress, suggesting that Nrf2 contributes to the anti-atherosclerosis response via the ARE. To gain further insight into the roles that Nrf2 plays in the development of atherosclerosis, we examined how Nrf2 regulates gene expression in response to anti-atherogenic laminar flow (L-flow) or pro-atherogenic oscillatory flow (O-flow). Exposure of human aortic endothelial cells (HAECs) to L-flow, but not to O-flow, induced the expression of cytoprotective genes, such as NAD(P)H quinone oxidoreductase 1 (NQO1) by 5-fold and heme oxygenase-1 by 8-fold. The critical contribution of Nrf2 to the expression induced by L-flow was ascertained in siRNA-mediated knock-down experiments. Two cyclooxygenase-2 (COX-2) specific inhibitors attenuated Nrf2 nuclear accumulation in the acute phase of L-flow exposure. A downstream product of COX-2, 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2), activated the Nrf2 regulatory pathway in HAECs through binding to the cysteines of Keap1. These results demonstrate that 15d-PGJ2 is essential for L-flow to activate Nrf2 and induce anti-atherosclerotic gene expression. Whereas both L-flow and O-flow induced the nuclear accumulation of Nrf2 to comparable levels, chromatin immunoprecipitation analysis revealed that Nrf2 binding to the NQO1 ARE was significantly diminished in the case of O-flow compared with that of L-flow. These results suggest that O-flow inhibits Nrf2 activity at the DNA binding step, thereby suppressing athero-protective gene expression and hence predisposing the blood vessels to the formation of atherosclerosis.

Aorta↗

PubMed2005

A simple method of isolating mouse aortic endothelial cells.

In the study of vascular biology, analyses of endothelial cells (EC) and smooth muscle cells (SMC) are very important. The mouse is a critical model for research, however, the isolation of primary EC from murine aorta is considered difficult. Previously reported procedures for the isolation of EC have required magnetic beads, or Fluorescence Activated Cell Sorting (FACS) to purify the cells. In addition, these procedures were applied to the heart, eyeball, or lung, not the aorta. Therefore we developed a simple method of isolating EC or SMC from the murine aorta without the need for any special equipment. To verify the purity of the cell culture, we performed both an immunofluorescence study and a DNA microarray analysis. The immunofluorescence study demonstrated specific expression of PECAM-1 in isolated EC cultures. In contrast, the isolated SMC didn't exhibit PECAM-1, but rather, smooth muscle actin. The DNA microarray analysis demonstrated the expression of EC (16 genes) or SMC (5 genes) specific genes in each cell. This is due to the fact that pure EC or SMC can be isolated from the aorta, without the use of any special equipment. These results suggest that this method should be particularly useful for vascular biological research.

Animals↗

PubMed2004

Effect on endothelial cell gene expression of shear stress, oxygen concentration, and low-density lipoprotein as studied by a novel flow cell culture system.

A new cell culture system has been developed that reflects the vascular microenvironment. By means of this system the cultured cells are exposed not only to shear stress by the circulating culture medium, but also to an oxygen concentration gradient and certain critical blood components such as low-density lipoprotein (LDL) and monocytes. DNA microarray analysis was performed for human umbilical vein endothelial cells cultured in this system in the absence and presence of laminar flow at a low shear stress, 0.2 dyn/cm(2). In addition to shear stress, either an oxygen concentration gradient, or LDL (1 mg/ml), or both were applied. Many Nrf-2-regulating genes, such as heme oxygenase 1, NAD(P)H quinone oxidoreductase 1, solute carrier family 7 No. 11, and glutamate-cysteine ligase modifier subunit, were induced by laminar flow at very low shear stress regardless of the additional conditions. Certain genes were specifically affected by exposure to the oxygen gradient and/or LDL under shear stress, but the degree was very low. These results suggest that shear stress is the most critical factor affecting gene expression in endothelial cells and that Nrf-2-regulating proteins may contribute to protecting endothelial cells against other vascular stress. This system should provide highly relevant and useful information about both vascular physiology and pathology, in the latter on such urgent matters as the specific steps involved in atherogenesis.

Base Sequence↗

PubMed2003

A novel method of following oxidation of low-density lipoprotein using a sensitive fluorescent probe, diphenyl-1-pyrenylphosphine.

Diphenyl-1-pyrenylphosphine (DPPP), which reacts with lipid hydroperoxide stoichiometrically to yield a fluorescent product DPPP oxide (DPPP=O) and the corresponding hydroxide, was used as a fluorescent probe for lipid peroxidation in low-density lipoprotein (LDL). DPPP was successfully incorporated into LDL using the dispersion reagent Pluronic F-127. Incorporation of DPPP into LDL was confirmed by gel filtration chromatography. Reaction of DPPP with hydroperoxide within an LDL particle was examined by monitoring the increase in fluorescence intensity of the LDL. It was found that lipid-soluble hydroperoxides such as methyl linoleate hydroperoxide preferably reacted with DPPP, whereas hydrogen peroxide did not. Fluorescence was increased at the early stages in the oxidation of DPPP-labeled LDL by an azo radical initiator or human neutrophils. LDL, which was labeled with DPPP or DPPP=O, was taken up by cells such as THP-1-derived macrophages and human umbilical vein endothelial cells. The fluorescence of DPPP=O could be observed in cells using fluorescence microscopy equipped with a cooled charge coupled device camera in a nondestructive manner. The present study shows that DPPP is a sensitive, selective, and quantitative probe for monitoring LDL oxidation and visualizing intracellular oxidation.

Cell Line↗