Peroxynitrite augments fibroblast-mediated tissue remodeling via myofibroblast differentiation

Irreversible airflow limitation in asthma is associated with airway remodeling in which the differentiation of fibroblasts to myofibroblasts plays a pivotal role. In asthmatic airways, excessive production of reactive nitrogen species (RNS) has been observed. The aim of this study is to evaluate whether peroxynitrite, one of the RNS, can affect the differentiation of fibroblasts to myofibroblasts. Human fetal lung fibroblasts were treated with various concentrations of authentic peroxynitrite or a peroxynitrite donor 3-morpholinosydnonimine hydrochloride (SIN-1), and the expressions of alpha-smooth muscle actin (alpha-SMA) and desmin, markers of myofibroblast differentiation, were evaluated. The releases of transforming growth factor-beta(1) (TGF-beta(1)) and ECM proteins including fibronectin and collagen I were assessed. To clarify the mechanism in this differentiation, the effect of anti-TGF-beta antibody or NF-kappaB inhibitors on the alpha-SMA expression and ECM production was assessed. Peroxynitrite and SIN-1 significantly augmented the alpha-SMA expression compared with control in a concentration-dependent manner (P < 0.01 and P < 0.05, respectively). Peroxynitrite significantly increased desmin and TGF-beta(1) production (P < 0.01). Peroxynitrite enhanced the translocation of NF-kappaB into the nucleus confirmed by immunocytostaining and immunoblotting. Peroxynitrite-augmented alpha-SMA expression was blocked by NF-kappaB inhibitors, MG132 and caffeic acid phenethyl ester (CAPE), and anti-TGF-beta antibody. CAPE completely inhibited the peroxynitrite-augmented TGF-beta(1) release. The production of fibronectin and collagen I was significantly increased by peroxynitrite (P < 0.01) and inhibited by anti-TGF-beta antibody. These results suggest that RNS can affect the differentiation to myofibroblasts and excessive ECM production via a NF-kappaB-TGF-beta(1)-dependent pathway.     

Protein phosphatase 2A regulatory subunit Bbeta promotes MAP kinase-mediated migration of A431 cells.

BACKGROUND/AIMS: Phosphatases are involved in regulation of MAP kinase (MAPK). A431 cells migrate on collagen after EGF stimulation using MAPK. To clarify the involvement of PP2A in this MAPK-dependent migration, the expression of an isoform of the B regulatory subunit was inhibited. METHODS: An antisense sequence corresponding to Bbeta cDNA was transfected into A431 cells. Their migratory activity on collagen was examined using Transwell, and MAPK phosphorylation and phosphatase activity were measured, and the results were compared with those obtained with mock-transfected cells. RESULTS: Antisense-transfected cells showed less Bbeta protein and phosphatase activity than mock-transfected controls. Migration of antisense-transfected cells showed a low response to EGF. The response of MAPK phosphorylation of antisense-transfected cells to EGF stimulation and adhesion to collagen in the presence or absence of EGF were markedly decreased. Phosphatase activity of PP2A-Bbeta also did not respond to EGF, collagen or EGF plus collagen, and remained at low levels. CONCLUSION: These results suggested that PP2A-Bbeta promotes cell migration through the MAPK cascade.     

Intraluteal release of angiotensin II and progesterone in vivo during corpora lutea development in the cow: effect of vasoactive peptides.

The newly formed corpus luteum (CL) develops rapidly and has the features of active vascularization and mitosis of steroidogenic cells. Such local mechanisms must be strictly regulated by the complex relationship between angiogenic growth factors and vasoactive peptides such as angiotensin (Ang) II, atrial natriuretic peptide (ANP), and endothelin (ET)-1. Thus, the objective of the present study was to determine 1) the changes in vasoactive peptides and progesterone (P) concentrations within the developing CL, along with the changes in concentration in ovarian venous plasma (OVP) and jugular venous plasma (JVP) in the cow, 2) the effects of CL exposure to vasoactive peptides on Ang II and P secretion, and 3) the expression of mRNA for ANP type C receptor in the bovine CL and endothelial cells (ETC) from bovine developing CL. A microdialysis system (MDS) was surgically implanted into multiple CL of six cows on Day 3 after a GnRH injection that induced superovulation, and a catheter was simultaneously inserted into the ovarian vein. The Ang II concentration in OVP was higher than that in JVP throughout the experiment, while the intraluteal release of Ang II was stable. During the experimental period, the concentrations of other vasoactive peptides (ANP and ET-1) showed no clear changes in plasma and were below detectable levels in the MDS perfusate. Exposure of CL to Ang II using the MDS stimulated P release, while exposure to ANP enhanced Ang II release within the developing CL. However, ET-1 had no effect on either P or Ang II release. The expression of mRNA for ANP type C receptor was mainly observed in early CL and ETC. The results suggest that the ET-Ang-ANP system in the preovulatory follicle switches to an Ang-ANP system to enhance both the angiogenesis and steroidogenesis that are actively occurring in developing CL.     

Genetic analysis of the chromosome of alkaliphilic Bacillus halodurans C-125

Seventeen Sse8387I linking clones isolated from the chromosome of Bacillus halodurans C-125 for the purpose of constructing a physical map were sequenced and analyzed by comparison with the BSORF database and the nonredundant protein databank. The orientations of Sse8387I or AscI linking clones serving to join adjacent fragments were determined by southern blot analysis using specific DNA probes. One-third of the open reading frames (ORFs) identified in the Sse8387I linking clones showed no significant similarity to any protein so far reported. The ORFs showing significant similarities to those of Bacillus subtilis were mapped in the chromosome of strain C-125, and the locations of the putative genes on the map were not well conserved between B. halodurans C-125 and B. subtilis. Received: March 26, 1999 / Accepted: April 27, 1999     

Discovery of novel scaffolds for γ-secretase modulators without an arylimidazole moiety

Gamma-secretase modulators (GSMs) selectively inhibit the production of amyloid-β 42 (Aβ42) and may therefore be useful in the management of Alzheimer’s disease. Most heterocyclic GSMs that are not derived from nonsteroidal anti-inflammatory drugs contain an arylimidazole moiety that potentially inhibits cytochrome P450 (CYP) activity. Here, we discovered imidazopyridine derivatives that represent a new class of scaffold for GSMs, which do not have a strongly basic end group such as arylimidazole. High-throughput screening identified 2-methyl-8-[(2-methylbenzyl)oxy]-3-(pyridin-4-yl)imidazo[1,2-a]pyridine (3a), which inhibited the cellular production of Aβ42 (IC₅₀?=?7.1?µM) without changing total production of Aβ. Structural optimization of this series of compounds identified 5-[8-(benzyloxy)-2-methylimidazo[1,2-a]pyridin-3-yl]-2-ethylisoindolin-1-one (3m) as a potent inhibitor of Aβ42 (IC₅₀?=?0.39?µM) but not CYP3A4. Further, 3m demonstrated a sustained pharmacokinetic profile in mice and sufficiently penetrated the brain.     

Effects of LPA1 and LPA6 on the regulation of colony formation activity in colon cancer cells treated with anticancer drugs

Lysophosphatidic acid (LPA) is a simple physiological lipid and exhibits a variety of cellular responses via the activation of G protein-coupled transmembrane LPA receptors (LPA receptor-1 (LPA₁) to LPA₆). The aim of our study was to investigate effects of LPA receptors on soft agar colony formation in colon cancer cells treated with anticancer drugs. DLD1 cells were treated with fluorouracil (5-FU) or cisplatin (CDDP) for at least six months (DLD-5FU and DLD-CDDP cells, respectively). LPAR1 gene expression was markedly elevated in DLD-5FU cells. In contrast, DLD-CDDP cells showed the high expression of LPAR6 gene. In colony formation assay, DLD-5FU cells formed markedly large-sized colonies, while no colony formation was observed in DLD1 and DLD-CDDP cells. The large-sized colonies formed in DLD-5FU cells were suppressed by LPA₁ knockdown. In contrast, LPA₆ knockdown increased the size of colonies. In addition, DLD-5FU cells were further treated with CDDP for three months (DLD-C-F cells). DLD-CDDP cells were also treated with 5-FU (DLD-F-C cells). DLD-C-F cells formed large-sized colonies, but not DLD-F-C cells, correlating with LPAR1 and LPAR6 gene expression levels. These results suggest that LPA₁ and LPA₆ may regulate the colony formation activity in DLD1 cells treated with anticancer drugs.