Yoshinori Kamisaki

A novel PPARγ gene therapy to control inflammation associated with inflammatory bowel disease in a murine model

BACKGROUND & AIMS Peroxisome proliferator-activated receptor gamma (PPAR gamma) is one of the nuclear receptors that plays a central role in adipocyte differentiation and insulin sensitivity. PPAR gamma has also recently been recognized as an endogenous regulator of intestinal inflammation. However, its levels are decreased during chronic inflammation in human and mice, thus limiting PPAR gamma ligand therapy during established disease. We sought to determine whether this decrease in PPAR gamma could be counteracted by a gene therapy approach. METHODS We characterized PPAR gamma levels in experimental colitis associated with dextran sodium sulfate administration to mice. In this model, the therapeutic benefits of PPAR gamma gene therapy using a replication-deficient adenovirus vector expressing PPAR gamma (Ad-PPAR gamma) was assessed. RESULTS PPAR gamma protein levels were decreased in whole colonic tissue, lamina propria lymphocytes, and peritoneal exudate cells during the course of colitis. PPAR gamma gene delivery using Ad-PPAR gamma restored responsiveness to a PPAR gamma ligand, resulting in marked amelioration of tissue inflammation associated with the colitis, which included attenuation of intercellular adhesion molecule-1, cyclooxygenase-2 and tumor necrosis factor-alpha expression. CONCLUSIONS Our results suggest that gene delivery of PPAR gamma can be used to restore and/or enhance endogenous anti-inflammatory processes that are normally operative in mammalian tissues such as in the colon.

Peroxisome Proliferator-activated Receptor γ-mediated Regulation of Neural Stem Cell Proliferation and Differentiation

Peroxisome proliferator-activated receptor γ (PPARγ) plays an important role in insulin sensitivity, tissue homeostasis, and regulating cellular functions. We found high-level expression of PPARγ in embryo mouse brain and neural stem cells (NSCs), in contrast to extremely low levels in adult mouse brain. Here, we show that PPARγ mediates the proliferation and differentiation of murine NSCs via up-regulation of the epidermal growth factor receptor and activation of the ERK pathway. Cell growth rates of NSCs prepared from heterozygous PPARγ-deficient mouse brains, PPARγ-RNA-silenced NSCs, and PPARγ dominant-negative NSCs were significantly decreased compared with those of wild-type NSCs. Physiological concentrations of PPARγ agonists, rosiglitazone and pioglitazone, stimulated NSC growth, whereas antagonists caused cell death in a concentration-dependent manner via activation of the caspase cascade. The stimulation of cell growth by PPARγ was associated with a rapid activation of the ERK pathway by phosphorylation and up-regulation of epidermal growth factor receptor and cyclin B protein levels. In contrast, activation of PPARγ by agonists inhibited the differentiation of NSCs into neurons. The inhibition of differentiation was associated with an activation of STAT3. These data indicate that PPARγ regulates the development of the central nervous system during early embryogenesis via control of NSC proliferation.

Histone H1.2 is a substrate for denitrase, an activity that reduces nitrotyrosine immunoreactivity in proteins

Several reports have described an activity that modifies nitrotyrosine-containing proteins and their immunoreactivity to nitrotyrosine Abs. Without knowing the product of the reaction, this new activity has been called a “denitrase.” In those studies, some nonspecific proteins, which have multiple tyrosine residues, e.g., albumin, were used as a substrate. Therefore, the studies were based on an unknown mechanism of reaction and potentially a high background. To solve these problems, one of the most important things is to find a more suitable substrate for assay of the enzyme. We developed an assay strategy for determining the substrate for denitrase combining 2D-gel electrophoresis and an on-blot enzyme assay. The resulting substrate from RAW 264.7 cells was Histone H1.2, an isoform protein of linker histone. Histone H1.2 has only one tyrosine residue in the entire molecule, which ensures the exact position of the substrate to be involved. It has been reported that Histones are the most prominent nitrated proteins in cancer tissues. It was also demonstrated that tyrosine nitration of Histone H1 occurs in vivo. These findings lead us to the idea that Histone H1.2 might be an intrinsic substrate for denitrase. We nitrated recombinant and purified Histone H1.2 chemically and subjected it to an on-blot enzyme assay to characterize the activity. Denitrase activity behaved as an enzymatic activity because the reaction was time dependent and was destroyed by heat or trypsin treatment. The activity was shown to be specific for Histone H1.2, to differ from proteasome activity, and to require no additional cofactors.

PPARγ ligands inhibit nitrotyrosine formation and inflammatory mediator expressions in adjuvant-induced rheumatoid arthritis mice

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor, whose activation has been linked to several physiologic pathways including those related to the regulation of insulin sensitivity. Here, we investigate effects of PPARgamma specific ligands, rosiglitazone and pioglitazone, on formation of nitrotyrosine and increased expression of inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 and intercellular adhesion molecule-1 (ICAM-1) in adjuvant-induced murine arthritis. Administration of rosiglitazone or pioglitazone (30 mg/kg, p.o.) significantly inhibited the adjuvant-induced increase in formation of nitrotyrosine and expression of iNOS on both ankle and temporomandibular joints. Rosiglitazone also inhibited the adjuvant-induced expression of M30 positive cells, as a marker of apoptosis, in the joint tissues. In addition, treatment with rosiglitazone or pioglitazone (30 microM) inhibited lipopolysaccharide plus tumor necrosis factor (TNF)-alpha-induced protein expression of iNOS, cyclooxygenase-2, ICAM-1 and nitrotyrosine formation in RAW 264 cells, a murine macrophage-like cell line. Rosiglitazone or pioglitazone inhibited increase in phosphorylated I-kappaB (pI-kappaB) expression, as an index of activation of nuclear factor (NF)-kappaB, in both joint tissues and RAW264 cells. Furthermore, in PPARgamma-transfected HEK293 cells, rosiglitazone inhibited the TNF-alpha-stimulated response using NF-kappaB-mediated transcription reporter assay. These results indicate that PPARgamma ligands may possess anti-inflammatory activity against adjuvant-induced arthritis via the inhibition of NF-kappaB pathway.

Presynaptic α2 adrenoceptors inhibit glutamate release from rat spinal cord synaptosomes

Abstract: The presynaptic regulation of amino acid release from nerve terminals was investigated using synaptosomes prepared from the rat spinal cord. The basal releases of endogenous glutamate (Glu), aspartate (Asp), and γ‐amino‐butyric acid (GABA) were 34.6, 21.5, and 10.0 pmol/min/mg of protein, respectively. Exposure to a depolarizing concentration of KCl (30 mM) evoked 2.7‐, 1.5‐, and 2.9‐fold increases in Glu, Asp, and GABA release, respectively. Clonidine reduced the K+‐evoked overflow of Glu to 56% of the control overflow with a potency (IC50) of 17 nM, but it did not affect K+‐evoked overflow of Asp, GABA, and their basal releases. Similarly, noradrenaline inhibited the K+‐evoked overflow of Glu, although phenylephrine and isoproterenol showed no effect. The inhibitory effect of clonidine was counteracted by α2‐adrenoceptor antagonists, rauwolscine, yohimbine, and idazoxan, regardless of the imidazoline structures. Because Glu is considered a neurotransmitter of primary afferents that transmit both nociceptive and nonnociceptive stimuli in the spinal cord, these data suggest that part of Glu release may be regulated by the noradrenergic system through α2 adrenoceptors localized on the primary afferent terminals.

Leukotriene B4 and lipoxin A4 are regulatory signals for neural stem cell proliferation and differentiation

Leukotrienes (LTs) and lipoxins (LXs) are lipid mediators that play a key role in regulating acute inflammatory responses. Their roles in neural stem cell (NSC) functions are of interest. We showed here that LTB4 and LXA4 regulated proliferation and differentiation of murine NSCs that were isolated from embryo brains. Proliferation of NSCs was stimulated by LTB4 (3 to 100 nM) and blocked by receptor antagonist (IC50=2.7 µM). In contrast, LXA4, and its aspirin‐triggered‐15‐epi‐LXA4 stable analog attenuated growth of NSCs at as little as 1 nM. Both lipoxygenase (LOX) inhibitors and LTB4 receptor antagonists caused apoptosis and cell death. Gene chip analysis revealed that growth‐related gene expressions such as epidermal growth factor (EGF) receptor, cyclin E, p27, and caspase 8 were tightly regulated by LTB4; LXA4 gave the opposite gene expressions. In addition to proliferation, LTB4 induced differentiation of NSCs into neurons as monitored by neurite outgrowth and MAP2 expression. These results indicate for the first time that LTB4 and LXA4 directly regulate proliferation and differentiation of NSCs, suggesting these new pathways may be useful in restoring stem cells.—Wada, K., Arita, M., Nakajima, A., Katayama, K., Kudo, C., Kamisaki, Y., Serhan, C. N. Leukotriene B4 and lipoxin A4 are regulatory signals for neural stem cell proliferation and differentiation. FASEB J. 20, 1785–1792 (2006)

Binding of [3H]p-aminoclonidine to two sites, α2-adrenoceptors and imidazoline binding sites: distribution of imidazoline binding sites in rat brain

Binding sites labeled by [3H]p-aminoclonidine [( 3H]PAC) were investigated by the competitive analysis with imidazoline and non-imidazoline derivatives. Phenylethylamine derivatives displaced only the part of specific sites for [3H]PAC, which was considered as alpha 2-adrenoceptor, whereas imidazoline derivatives, such as clonidine and tolazoline, competed for a further specific binding of [3H]PAC to the non-adrenergic sites, in addition to the alpha 2-adrenoceptor. Because the non-adrenergic sites were specific for the imidazoline structure, they were termed imidazoline sites. The imidazoline sites were not distributed uniformly among rat brain regions. In striatum, hippocampus and medulla oblongata, they occupied 39.6, 33.0 and 36.5% of the specific binding of [3H]PAC, respectively. Saturation isotherms revealed that Kd and Bmax of imidazoline sites for [3H]PAC were 3.09 +/- 0.59 nM, 27.4 +/- 1.7 fmol/mg protein and 2.23 +/- 0.29 nM, 21.0 +/- 1.5 fmol/mg protein in striatum and hippocampus, respectively. Because imidazoline binding sites also displayed weak affinities for imidazole compounds, such as histamine and cimetidine, the imidazoline site may be a subtype of histamine H2-receptor.

A new gastric ulcer model induced by ischemia-reperfusion in the rat: Role of leukocytes on ulceration in rat stomach

A new model of gastric ulcer involving damage to the muscularis mucosae was developed by clamping the celiac artery in rat to induce ischemia-reperfusion (I-R) injury. Although erosions with falling off of the gastric mucosa were observed immediately, 24 and 36 hours after the I-R, gastric ulcers involving the injury of muscularis mucosae were observed in the area of gastric glands at 48 and 72 hours after initiation of injury. Administration of omeprazol, a proton pump inhibitor, or pentoxifylline, an anti-leukocyte drug, just after the initiation of injury significantly decreased the total area of ulcers at 72 hours. A combination of omeprazol and pentoxifylline was more effective than either drug alone. An anti-leukocyte adhesion molecule (anti-CD18 antibody) also showed significant inhibitory effect on the development of ulcers at 72 hours and the infiltration of leukocytes into both submucosa and mucosa. These results indicate that in our model, gastric acid together with leukocytes contribute to the development of ulcers following erosions. This model may be used to investigate the mechanisms of the development of gastric ulcer and evaluate antiulcer drugs in a preclinical setting.

Involvement of a periodontal pathogen, Porphyromonas gingivalis on the pathogenesis of non-alcoholic fatty liver disease

BackgroundNon-alcoholic fatty liver disease (NAFLD) is a hepatic manifestation of metabolic syndrome that is closely associated with multiple factors such as obesity, hyperlipidemia and type 2 diabetes mellitus. However, other risk factors for the development of NAFLD are unclear. With the association between periodontal disease and the development of systemic diseases receiving increasing attention recently, we conducted this study to investigate the relationship between NAFLD and infection with Porphyromonas gingivalis (P. gingivalis), a major causative agent of periodontitis.MethodsThe detection frequencies of periodontal bacteria in oral samples collected from 150 biopsy-proven NAFLD patients (102 with non-alcoholic steatohepatitis (NASH) and 48 with non-alcoholic fatty liver (NAFL) patients) and 60 non-NAFLD control subjects were determined. Detection of P. gingivalis and other periodontopathic bacteria were detected by PCR assay. In addition, effect of P. gingivalis-infection on mouse NAFLD model was investigated. To clarify the exact contribution of P. gingivalis-induced periodontitis, non-surgical periodontal treatments were also undertaken for 3 months in 10 NAFLD patients with periodontitis.ResultsThe detection frequency of P. gingivalis in NAFLD patients was significantly higher than that in the non-NAFLD control subjects (46.7% vs. 21.7%, odds ratio: 3.16). In addition, the detection frequency of P. gingivalis in NASH patients was markedly higher than that in the non-NAFLD subjects (52.0%, odds ratio: 3.91). Most of the P. gingivalis fimbria detected in the NAFLD patients was of invasive genotypes, especially type II (50.0%). Infection of type II P. gingivalis on NAFLD model of mice accelerated the NAFLD progression. The non-surgical periodontal treatments on NAFLD patients carried out for 3 months ameliorated the liver function parameters, such as the serum levels of AST and ALT.ConclusionsInfection with high-virulence P. gingivalis might be an additional risk factor for the development/progression of NAFLD/NASH.

Sensitive determination of nitrotyrosine in human plasma by isocratic high-performance liquid chromatography

A highly sensitive and simple isocratic high-performance liquid chromatographic method was developed for determination of 3-nitrotyrosine in human plasma with precolumn derivatization with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole. The precision of the method was satisfactory (coefficient of variation 4.8%), and the detection limit was established at 0.1 pmol of 3-nitrotyrosine allowing the determination at the level of 6 pmol/ml in human plasma. The recoveries of 3-nitrotyrosine and alpha-methyltyrosine, an internal standard, were 89.3 +/- 7.1 and 85.7 +/- 7.6%, respectively. The 3-nitrotyrosine level was 31 +/- 6 pmol/ml (mean +/- S.D., n = 9) in plasma from healthy volunteers. Since 3-nitrotyrosine is a stable product of peroxynitrite, an oxidant formed by a reaction of nitric oxide and superoxide radicals, the measurement of its plasma concentration may be useful as a marker of nitric oxide-dependent oxidative damage.