Characterization of aortic aneurysms in cardiovascular disease patients harboring Porphyromonas gingivalis.

OBJECTIVE: Porphyromonas gingivalis was recently shown to cause intimal hyperplasia in a mouse model by a novel cholesterol-independent mechanism, suggesting to be a pathogen-specific feature of cardiovascular diseases. The aim of this study was to characterize the clinical and histopathological features of aortic aneurysms in cardiovascular disease patients harboring oral P. gingivalis. SUBJECT AND METHODS: Aortic aneurysm specimens were collected from 76 Japanese patients who underwent surgery, of whom dental plaque specimens were also collected from 31 patients. Bacterial DNA was extracted from each specimen to detect P. gingivalis by polymerase chain reaction. Histopathological analyses of the aortic aneurysm specimens, including immunohistochemical staining for embryonic myosin heavy chain isoform (SMemb) and S100 calcium-binding protein A9 (S100A9), were also performed. RESULTS: The number of aneurysms occurring in the distal aorta was significantly higher in subjects positive for P. gingivalis in dental plaque compared with those who were negative. The expressions of S100A9 and SMemb were also significantly greater in the subjects positive for P. gingivalis in dental plaque. On the other hand, there were no significant differences in adipocellular accumulation between the groups. CONCLUSIONS: These results suggest that aortic aneurysms in patients harboring oral P. gingivalis have greater expression of S100A9 and proliferative smooth muscle cells, which was different from the present patients without oral P. gingivalis.

Activation of peroxisome proliferator-activated receptor gamma suppresses mast cell maturation involved in allergic diseases.

BACKGROUND: Mast cells play a central role in allergic and inflammatory diseases. Several reports indicated role of peroxisome proliferator-activated receptor gamma (PPARgamma) on mast cell function. However, there is no report about the role of PPARgamma on differentiation of mast cells from the progenitors. In this study, we investigated the role of PPARgamma in regulating bone marrow-derived mast cell maturation and the therapeutic implications for mast cell-related diseases such as atopic or contact dermatitis. METHODS: We used in vitro cell culture system for mast cell differentiation from bone marrow-progenitors using specific ligands and lentiviral-mediated short hairpin RNA of PPARgamma, and in vivo murine dermatitis models. RESULTS: Activation of PPARgamma inhibited the maturation of bone marrow progenitors into connective tissue-type mast cells (CTMCs) through up-regulation of GATA-4 and GATA-6 resulting in a decrease in expression of histidine decarboxylase and mast cell histamine content. In comparison, the differentiation of bone marrow progenitors into CTMCs was significantly accelerated by the knockdown of PPARgamma expression by lentiviral-mediated short hairpin RNA. Peroxisome proliferator-activated receptor gamma ligand administration to mice inhibited the maturation of mast cells resulting in attenuation of atopic and contact dermatitis via diminishment of the number of mature mast cells. CONCLUSION: Our results indicate that PPARgamma is one of master regulators on mast cell maturation and potentially useful for the therapy in various disorders involving mast cell activation.

Intracranial dural arteriovenous fistulas with retrograde cortical venous drainage: assessment with cerebral blood volume by dynamic susceptibility contrast magnetic resonance imaging.

BACKGROUND AND PURPOSE: Retrograde cortical venous drainage (RCVD) is the most major risk factor for aggressive behavior of intracranial dural arteriovenous fistulas (DAVF). The purpose of this study was to assess the efficacy of relative cerebral blood volume (rCBV) map for RCVD in patients with DAVF. METHODS: Ten patients with angiographically proven DAVF with RCVD, 2 reference patients with DAVF without RCVD, and 10 control subjects underwent examinations with dynamic susceptibility contrast (DSC)-MR imaging. Four patients with DAVF with unilateral RCVD were evaluated, before and after treatment. The calculation of mean rCBV ratio was performed on a hemispheric basis. The mean rCBV ratio was defined as the value on one side (higher value side) divided by that on the other side (lower value side). RESULTS: In all patients with DAVF with RCVD, the rCBV map showed an increase in rCBV of the angiographically proved affected hemisphere. In 2 reference patients with DAVF without RCVD and all control subjects, the rCBV map showed no increase of rCBV. The mean rCBV ratio in patients with DAVF with RCVD was significantly higher than that of control subjects (P = .0002). Treatment response for RCVD was indicated by a decrease of CBV on the rCBV map and by a decrease of 22% in the mean rCBV ratio. CONCLUSIONS: Increased rCBV by DSC-MR correlated with RCVD in patients with DVAF. The assessment with rCBV for RCVD may be more quantitative than that with angiogram.

Lipopolysaccharide-induced increase in plasma nitrotyrosine concentrations in rats.

Since the production of peroxynitrite may contribute to the pathophysiology of endotoxemia or sepsis, the quantities of the produced peroxynitrite were evaluated in rats after lipopolysaccharide (LPS) treatment by measuring plasma nitrotyrosine concentrations with a new method. The intraperitoneal administration of LPS caused a persistent increase in plasma nitrotyrosine concentrations, which reached a maximum with 6-fold level of the base line (105 pmol ml-1) at 24 h and gradually declined to 3-fold level of the base line at 7 days. However, plasma concentrations of nitrite and nitrate peaked at 18 h, returning to base line within 48 h. The effect of LPS on the increase in plasma concentration of nitrotyrosine was dose-dependent and consistent with that of nitrite and nitrate concentrations. On the other hand, intravenous injection of nitrotyrosine revealed a rapid clearance with a plasma half-life of 1.67 h. These results indicate that the elevation of plasma nitrotyrosine concentrations may persist for more than a week after LPS treatment, and that the determination of plasma nitrotyrosine concentrations may be useful to detect the previous peroxynitrite-dependent oxidative damages.

Effects of cigarette smoke on nitric oxide-induced increase in cyclic GMP in nerve terminals of rat cerebral cortex.

Since nitric oxide (NO) has been widely accepted as a novel neuromodulator, which activates soluble forms of guanylate cyclase to increase in guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels, the effect of water-soluble substance in cigarette smoke on cyclic GMP levels were investigated using nerve terminals prepared from rat cerebral cortex. Although the smoke-substance itself failed to affect cyclic GMP levels in the synaptosomes, the smoke-substance significantly inhibited the increases in cyclic GMP levels induced by NO donors. The blocking effect of the smoke-substance was inhibited by concomitant incubation with superoxide dismutase, but not with mannitol. In addition, the effect of smoke-substance was mimicked by products of the xanthine/xanthine oxidase system, but not by nicotine. The effect of smoke-substance was preserved at least 7 days after they were stored at room temperature. Therefore, these results suggest that the smoke-substance may possess long half-lives to produce the radicals which inactivate NO, and to inhibit the increase in cyclic GMP levels in nerve terminals. The interference with NO may explain the part of mechanism in effects of cigarette smoke on neuronal functions.

Frequent loss in chromosome 8p loci in liver cirrhosis accompanying hepatocellular carcinoma.

Most hepatocellular carcinoma (HCC) is preceded by liver cirrhosis, but the genetic changes involved in cirrhosis are not well understood. We therefore studied loss of heterozygosity (LOH) in cirrhotic and neoplastic foci in livers of 14 patients with HCC. The samples, microdissected from paraffin-embedded tissues, were analyzed using a polymerase-chain-reaction-based assay for dinucleotide repeat polymorphisms on 8p. Of the 14 cases, 13 showed constitutional heterozygosity for the microsatellite markers. In 7 (54%) of these 13 informative cases, LOH was detected in the primary HCC and, in these 7 doubly informative (informative and LOH-positive in primary HCC) cases, LOH was found in 16 (70%) of 23 liver cirrhotic foci. The pattern of 8p allelic loss was identical in each doubly informative tumor; however, some of the liver cirrhotic foci harbored an 8p loss identical to that seen in the primary HCC, some harbored a different 8p loss, and some did not harbor any 8p loss. The remaining 6 cases without LOH on 8p in HCC showed no 8p loss in any cirrhotic foci. Presumably HCC could develop from cirrhotic cells harboring 8p loss.

Loss of heterozygosity of the retinoblastoma gene in liver cirrhosis accompanying hepatocellular carcinoma.

Carcinogenesis is a multistep process. Most hepatocellular carcinoma (HCC) is preceded by liver cirrhosis, but the genetic changes involved in cirrhosis are not known well. The present study was conducted to evaluate aberration of the retinoblastoma (RB) gene in HCC and adjacent non-tumorous liver using 22 patients with chronic liver damage accompanying HCC. The specimens obtained by microdissection from paraffin-embedded tissues were analyzed using an assay based on the polymerase chain reaction for highly polymorphic nucleotide sequences of microsatellites in the RB gene. Out of 22 cases, 15 showed constitutional heterozygosity for the microsatellite markers. In 11 (73.3%) of these 15 informative cases, the primary HCC foci showed loss of heterozygosity (LOH). In 8 of these 11 doubly informative (informative and LOH-positive in primary HCC) cases, LOH was found in 20 (64.5%) of 31 microdissected non-tumorous foci. All of the non-tumorous foci showing RB loss were cirrhotic lesions but there were no foci of chronic hepatitis. The remaining 4 cases without LOH in HCC foci showed no LOH in non-tumorous lesions. In our study, LOH of the RB gene was frequently observed in liver cirrhosis surrounding tumor.

Nullification of a positive correlation between urinary contents of alpha1-microglobulin and ulinastatin with intracerebroventricularly administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium (MPP+) in mice.

Striatal dopamine contents in C57BL/6J mice were reduced at 24 h after intracerebroventricular (ICV) administration of 1-methyl-4-phenyl-1,2, 3,6-tetrahydropyridine (MPTP) or 1-methyl-4-phenylpyridinium (MPP+) in a dose-dependent manner. A dose of 1.8 microg MPP+ significantly (P < 0.05) suppressed the dopamine contents, whereas a similar dose of MPTP did not. A definite positive correlation between urinary contents of alpha1-microglobulin (alpha1M) and ulinastatin (UT) existed in normal mice. However, this correlation was nullified by ICV administration of 18 and 36 microg MPTP or 1.8 and 18 microg MPP+. With 1.8 microg MPTP, a positive correlation between urinary contents of alpha1M and UT was displayed. The urine volume, creatinine content, glomerular filtration rate, alpha1M and UT contents, and alpha1M/UT ratio of urine collected for 24 h post-ICV administration of MPTP or MPP+, were not statistically different from those of control mice. Our findings suggest that the central effects of MPP+, a neurotoxic metabolite of MPTP, nullify the positive correlation between urinary contents of alpha1M and UT without affecting renal functions.

Involvement of mitogen-activated protein kinase in peroxynitrite-induced cell death of human neuroblastoma SH-SY5Y cells.

We have investigated the activation of mitogen-activated protein kinase (MAP kinase) in relation to cell death induced by peroxynitrite in human neuroblastoma SH-SY5Y cells. Exposure of the cells to peroxynitrite caused transient increase in MAP kinase activity, and resulted in cell death. PD98059, a selective inhibitor of MAP kinase kinase, reduced peroxynitrite-induced cell death. These results suggest that the activation of MAP kinase may be involved in cell death induced by peroxynitrite.

Clonidine inhibition of potassium-evoked release of glutamate and aspartate from rat cortical synaptosomes.

Release of endogenous glutamic acid (Glu), aspartic acid (Asp) and gamma-aminobutyric acid (GABA) has been investigated using synaptosomes prepared from rat cerebral cortex. Exposure in superfusion to a depolarizing concentration of KCl (30 mM) evoked 3-, 2- and 2-fold increases in Glu, Asp and GABA release, respectively. More than 70% of Glu and Asp overflow were calcium-dependent, although 67% of the GABA overflow was calcium-independent. Clonidine inhibited the K(+)-evoked overflow of Glu and Asp in a concentration-dependent manner, but the GABA overflow was not inhibited. Clonidine inhibited K(+)-evoked Glu and Asp overflow to 40 and 30% of the control with a potency (IC50) of 11 and 36 nM, respectively. Similarly, norepinephrine inhibited the K(+)-evoked overflow of Glu and Asp, although phenylephrine and isoproterenol showed no effect. Rauwolscine, yohimbine and idazoxan counteracted the effects of clonidine on Glu and Asp overflow. The data suggest that the depolarization-evoked overflow of excitatory amino acids is regulated in an inhibitory fashion by alpha 2 adrenoceptors, which are located on the nerve terminals of Glu and Asp neurons in rat cortex.

Effects of taurine on depolarization-evoked release of amino acids from rat cortical synaptosomes.

Effects of taurine on endogenous aspartic acid (Asp), glutamic acid (Glu) and gamma-aminobutyric acid (GABA) release has been investigated using synaptosomes prepared from rat cerebral cortex. Although basal release of these amino acids was not affected, taurine inhibited KCl (30 mM)-evoked overflow of Asp, Glu and GABA in a concentration-dependent manner with potencies (IC50) of 1 microM, 0.8 microM and 5 nM, respectively. Taurine (10 microM) maximally inhibited K(+)-evoked Asp, Glu and GABA overflow by 28, 37 and 65%, respectively. Phaclofen (10 microM, a GABAB receptor antagonist), but not bicuculline (10 microM, a GABAA receptor antagonist), counteracted the inhibition of GABA overflow, although the inhibition of Asp and Glu overflow was not attenuated. These data suggest that taurine may inhibit GABA release through the activation of presynaptic GABAB autoreceptors and, at high concentration, also act on Asp- and Glu-nerve terminals to regulate release of excitatory amino acids in rat cortex.

Immunocytochemical localizations of cytosolic and mitochondrial glutamic oxaloacetic transaminase isozymes in rat brain.

The localizations of cytosolic (s-) and mitochondrial (m-) glutamic oxaloacetic transaminase (GOT) were examined by immunocytochemical methods using specific antibodies. Staining of s-GOT-like immunoreactivity was seen in periglomerular cells of the olfactory bulb, and basket, stellate cells of the cerebellum, and second layer cells of the neocortex. On the other hand, m-GOT-like immunoreactivity was found in mitral cells and glomerular regions of the olfactory bulb and deep Golgi cells of the cerebellum. These different distributions of s- and m-GOT isozymes suggest that these isozymes are available as markers of glutamergic or aspartergic neurons.

No enhancement by nitric oxide of glutamate release from P2 and P3 synaptosomes of rat hippocampus.

Effects of nitric oxide on glutamate (Glu) release in long-term potentiation (LTP) were investigated by superfusion of conventional (P2) and large (P3) synaptosomes prepared from the rat hippocampus. Basal releasing rates of endogenous Glu from P2 and P3 fractions were 103.6 and 85.2 pmol/min/mg protein, respectively. Exposure to a depolarizing concentration of KCl (30 mM) evoked 3.58- and 4.52-fold increases in releasing rates of Glu from P2 and P3 fractions, respectively. Although the perfusion with sodium nitroprusside (NP, 10(-3) M), a nitric oxide-releasing agent, failed to augment the K(+)-evoked releases of Glu from P2 and P3 synaptosomes, NP enhanced that from slices of the hippocampus by 39% without changing basal release. Similarly, 8-bromoguanosine 3':5'-cyclic monophosphate (10(-4) M) increased the K(+)-evoked release of Glu from slices by 30%, but not from either synaptosomes. When synaptosomes were prepared from the hippocampus which was pretreated with two trains of electrical field stimulation (100 Hz, 0.1 ms, for 2 s), K(+)-evoked releases of Glu from P2 and P3 synaptosomes were increased by 15% and 23%, respectively. Although nitric oxide is postulated to function as a retrograde messenger to maintain LTP, present results suggest that nitric oxide may not directly act upon nerve terminals to enhance glutamate release, but that interventions of glias and short neurons may be involved in the presynaptic mechanism of LTP.

Immunocytochemical localizations of cytosolic and mitochondrial glutamic oxaloacetic transaminase isozymes in rat retina as markers for the glutamate-aspartate neuronal system.

The localization of cytosolic (s) or mitochondrial (m) glutamic oxaloacetic transaminase (GOT) was examined in the rat retina by means of an indirect immunofluorescence method using antibodies specific for s- and m-GOT. The m-GOT-like immunoreactive structures were seen on the inner segments of the photoreceptor cells and other outer and inner plexiform layers. These structures were dot-like in appearance. Somas were not labeled. In contrast, s-GOT-like structures were found on the inner segments and inner fibers of the photoreceptor cells, numerous cell somas in the inner nuclear layer (horizontal, amacrine and bipolar cells), and ganglion cell layer (displaced amacrine cells) and inner plexiform layer. The difference in distribution between s- and m-GOT isozymes suggests that they may be useful as markers for glutamatergic and/or aspartinergic neurons.

Binding of [3H]p-aminoclonidine to two sites, alpha 2-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.

Immunocytochemical localizations of cytosolic and mitochondrial glutamic oxaloacetic transaminase isozymes in rat primary sensory neurons as a marker for the glutamate neuronal system.

The localization of cytosolic (s-) and mitochondrial (m-) glutamic oxaloacetic transaminase (GOT) was examined in the rat trigeminal, jugular and dorsal root ganglia by means of an indirect immunofluorescence method using antibodies specific for s- and m-GOT. Staining of s-GOT-like immunoreactivity was seen in giant, large, medium and small cells in these ganglia. On the other hand, m-GOT-like immunoreactivity was not seen in them. The distribution of GOT suggests that glutamate may be a transmitter released from primary sensory neurons.

Effects of histamine H2 receptor antagonists on acid secretion stimulated by imidazoline derivatives in isolated parietal cells.

Both the effect of imidazoline derivatives on acid secretion and the mechanism of this effect were studied in the parietal cells isolated from guinea pigs. Clonidine and tolazoline both stimulated the parietal cells to elevate the concentration of cyclic AMP and the accumulation of [14C]aminopyrine concentration dependently, although these imidazoline derivatives are known as alpha 2-adrenoceptor agonist or antagonist. These stimulatory effects were inhibited by famotidine, ranitidine and cimetidine, histamine H2 receptor antagonists. However, [3H]clonidine binding to the membrane preparations of parietal cells was not affected by these H2 antagonists and yohimbine but was inhibited by imidazoline derivatives. These results suggest that imidazoline derivatives may bind to the specific binding sites (different from H2 receptor or alpha 2-adrenoceptor) and stimulate the acid secretion of parietal cells with an increase of cyclic AMP, and that H2 antagonists may not only compete with the agonists for receptor binding but also interfere with the receptor adenylate cyclase system.

Beta-adrenergic regulation of amine precursor amino acid transport across the blood-brain barrier.

Beta-Adrenoceptor agonists were administered i.p. into rats and amino acid levels in brain and plasma were then determined to assess the effects on transport across the blood-brain barrier. Isoproterenol (10 mumol/kg) caused significant increases in aromatic amino acid (tyrosine, phenylalanine and tryptophan) levels in cerebral cortex and decreases in almost all amino acid concentrations in plasma. This effect of isoproterenol on brain tyrosine level was dose-dependent with an ED50 of 0.25 mumol/kg. Salbutamol (beta 2-adrenoceptor agonist, 10 mumol/kg) showed similar effects, but dobutamine (beta 1-adrenoceptor agonist, 50 mumol/kg) failed to increase brain amino acid levels. When 1-threo-3,4-dihydroxyphenylalanine (L-DOPA, 100 mumol/kg) was i.p. loaded, beta-adrenoceptor agonists promoted the transport of L-DOPA into brain without increasing the clearance rate of plasma L-DOPA. Moreover, significant increases in dopamine and its metabolites were observed in rat brain. These findings suggest that the transport of aromatic amino acids across the blood-brain barrier may be regulated through beta 2-adrenoceptors and that co-administration of beta 2-adrenoceptor agonists with L-DOPA may enhance the therapeutic efficacy of L-DOPA.

Inhibition of cyclic AMP accumulation by alpha 2-adrenoceptors in the rat cerebral cortex.

The effects of alpha 2-adrenoceptor agonist and antagonists on the accumulation of cyclic AMP were examined in rat cerebral cortex slices. Norepinephrine (10(-4) M) caused a 123 +/- 11% increase in the cyclic AMP concentration in the cortical slices, which was greater than the increase (89 +/- 7% increase) caused by isoproterenol (10(-4) M) alone. However, the cyclic AMP response to norepinephrine was completely inhibited by propranolol (10(-4) M), a beta-adrenoceptor antagonist. Yohimbine (10(-7)-10(-5) M), an alpha 2-adrenoceptor antagonist, intensified the cyclic AMP response to norepinephrine by 30%, whereas, clonidine, an alpha 2-adrenoceptor agonist, decreased the response. Treatment with reserpine (3.0 mg/kg) reduced the density of [3H]p-aminoclonidine binding sites (Bmax, 93.8 +/- 18.4 fmol/mg protein) compared to the density in non-treated rats (154.4 +/- 33.5 fmol/mg protein). The potentiating effect of yohimbine and the inhibitory effect of clonidine on the cyclic AMP response to norepinephrine were also reduced. These results suggest that alpha 2-adrenoceptors regulate the accumulation of cyclic AMP in the rat cerebral cortex in an inhibitory fashion. The results also suggest that the accumulation is mediated through beta-adrenoceptors and that this response is intensified by alpha 1-adrenoceptor stimulation.

Effect of cystathionine as a scavenger of superoxide generated from human leukocytes or derived from xanthine oxidase in vitro.

We studied the direct effects of cystathionine on human leukocyte-generated or xanthine-xanthine oxidase-derived superoxide radicals in vitro. Washed leukocyte suspensions (10(6) cells/ml) prepared from healthy male volunteers were stimulated with phorbol myristate acetate (1 mu M) or opsonized zymosan (1 mg/ml) to generate superoxide radicals, which were measured with a 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-alpha] pyrazin-3-one hydrochloride (MCLA)-chemiluminescence method. Cystathionine (30 mu M to 10 mM) significantly reduced superoxide radical-dependent chemiluminescence in the leukocyte system in a concentration-dependent manner. In addition, in the two different methods of determination of superoxide radicals (MCLA chemiluminescence and nitroblue tetrazolium reduction), cystathionine significantly scavenged the superoxide radicals derived from the xanthine-xanthine oxidase system. However, cystathionine did not inhibit the activity of xanthine oxidase during superoxide generation. On the other hand, cystathionine did not show a scavenging effect against hydroxyl radicals derived from Fe2+ -H2O2 on the erythrocyte membrane. These results indicate that cystathionine itself may possess a scavenging function against superoxide radicals rather than against hydroxyl radicals in vitro.