Gut Microbiota Influence the Development of Abdominal Aortic Aneurysm by Suppressing Macrophage Accumulation in Mice.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease characterized by dilated abdominal aorta. Immune cells have been shown to contribute to the development of AAA, and that the gut microbiota is associated with numerous diseases, including cardiovascular diseases, by regulating immune systems or metabolic pathways of the host. However, the interaction between the gut microbiota and AAA remains unknown. METHODS: Apolipoprotein E-deficient male mice were fed a high-cholesterol diet and divided into three groups: the control group was maintained under normal water (control group), the oral AVNM group was maintained under drinking water supplemented with ampicillin, vancomycin, neomycin, and metronidazole, and the i.p. AVNM group was injected AVNM intraperitoneally. After 1 week of pretreatment with antibiotics, these mice were administrated Ang II via subcutaneous osmotic pumps for 4 weeks and euthanized to evaluate AAA formation. RESULTS: Depletion of gut microbiota by oral AVNM ameliorated the incidence of AAAs (control group: 58.9% versus oral AVNM group: 28.6% versus i.p. AVNM group: 75.0%, P = 0.0005) and prevented death due to ruptured aneurysms (control group: 11% versus oral AVNM group: 0% versus i.p. AVNM group: 15%). Oral AVNM suppressed monocyte storage in the spleen, but not in other organs. Despite possessing a higher level of cholesterol, recruitment of monocytes into the suprarenal aorta was suppressed in the oral AVNM group. In AVNM drinking mice, NOD1 ligand, a kind of PRR ligands, increased the development of AAAs and accumulation of macrophages in the aortae. CONCLUSIONS: The gut microbiota plays a critical role in AAA formation. Therefore, regulation of the microbiota or the immune system can be a therapeutic approach for AAA.

CDP-ribitol prodrug treatment ameliorates ISPD-deficient muscular dystrophy mouse model.

Ribitol-phosphate modification is crucial for the functional maturation of α-dystroglycan. Its dysfunction is associated with muscular dystrophy, cardiomyopathy, and central nervous system abnormalities; however, no effective treatments are currently available for diseases caused by ribitol-phosphate defects. In this study, we demonstrate that prodrug treatments can ameliorate muscular dystrophy caused by defects in isoprenoid synthase domain containing (ISPD), which encodes an enzyme that synthesizes CDP-ribitol, a donor substrate for ribitol-phosphate modification. We generated skeletal muscle-selective Ispd conditional knockout mice, leading to a pathogenic reduction in CDP-ribitol levels, abnormal glycosylation of α-dystroglycan, and severe muscular dystrophy. Adeno-associated virus-mediated gene replacement experiments suggested that the recovery of CDP-ribitol levels rescues the ISPD-deficient pathology. As a prodrug treatment strategy, we developed a series of membrane-permeable CDP-ribitol derivatives, among which tetraacetylated CDP-ribitol ameliorated the dystrophic pathology. In addition, the prodrug successfully rescued abnormal α-dystroglycan glycosylation in patient fibroblasts. Consequently, our findings provide proof-of-concept for supplementation therapy with CDP-ribitol and could accelerate the development of therapeutic agents for muscular dystrophy and other diseases caused by glycosylation defects.

Metabolic alterations in plasma after laparoscopic sleeve gastrectomy.

Laparoscopic sleeve gastrectomy (LSG) is an important therapeutic option for morbidly obese patients. Although LSG promotes sufficient weight loss, how LSG changes plasma metabolites remains unclear. We assessed changes in plasma metabolite levels after LSG. We collected plasma samples from 15 morbidly obese Japanese patients before and 3 months after LSG. A total of 48 metabolites were quantified using capillary electrophoresis time-of-flight mass spectrometry-based metabolomic profiling. Branched chain amino acids, several essential amino acids, choline, 2-hydroxybutyric acid, 2-oxoisovaleric acid and hypoxanthine were significantly decreased after LSG. Tricarboxylic acid cycle metabolites, including citric acid, succinic acid and malic acid, were significantly elevated after LSG. This is the first report to show dynamic alterations in plasma metabolite concentrations, as assessed using capillary electrophoresis time-of-flight mass spectrometry, in morbidly obese patients after LSG. Our results might show how LSG helps improve obesity, in part through metabolic status changes, and propose novel therapeutic targets to ameliorate obesity.

Bioconversion of 5-deoxystrigol stereoisomers to monohydroxylated strigolactones by plants.

The bioconversion of 5-deoxystrigol (5DS) and 4-deoxyorobanchol (4DO), the simplest canonical strigolactones (SLs), into monohydroxylated SLs such as strigol, sorgomol and orobanchol was confirmed by administering of stable isotope-labeled substrates to hydroponically grown plants. Liquid chromatography-mass spectrometry analyses established that 5DS was stereoselectively converted into strigol and sorgomol by cotton (Gossypium hirsutum) and Chinese milk vetch (Astragalus sinicus), respectively. 4DO was converted into orobanchol by rice (Oryza sativa). However, the red bell pepper (Capsicum annuum), red clover (Trifolium pratense), and pea (Pisum sativum) negligibly converted 4DO into orobanchol. The red bell pepper converted ent-4DO into 2',8-bisepi-sorgomol. These results suggest that some plants generate orobanchol without passing through 4DO.

Regioselective and stereospecific hydroxylation of GR24 by Sorghum bicolor and evaluation of germination inducing activities of hydroxylated GR24 stereoisomers toward seeds of Striga species.

Bioconversion of GR24, the most widely used synthetic strigolactone (SL), by hydroponically grown sorghum (Sorghum bicolor) and biological activities of hydroxylated GR24 stereoisomers were studied. Analysis of extracts and exudates of sorghum roots previously fed with a racemic and diastereomeric mixture of GR24, using liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (MRM), confirmed uptake of GR24 and suggested its conversion to mono-hydroxylated products. Two major GR24 metabolites, 7-hydroxy-GR24 and 8-hydroxy-GR24, were identified in the root extracts and exudates by direct comparison of chromatographic behavior with a series of synthetic mono-hydroxylated GR24 analogues. Separate feeding experiments with each of the GR24 stereoisomers revealed that the hydroxylated products were derived from 2'-epi-GR24, an evidence of sterical recognition of the GR24 molecule by sorghum. Trans-4-hydroxy-GR24 isomers derived from all GR24 stereoisomers were detected in the exudates as minor metabolites. The synthetic hydroxy-GR24 isomers induced germination of Striga hermonthica in decreasing order of C-8>C-7>C-6>C-5>C-4. In contrast the stereoisomers having the same configuration of orobanchol, irrespective of position of hydroxylation, induced germination of Striga gesnerioides. The results confirm previous reports on structural requirements of SLs and ascribe a critical role to hydroxylation, but not to the position of the hydroxyl group in the AB part of the molecule, in induction of S. gesnerioides seed germination.

The molecular dynamics of crawling migration in microtubule-disrupted keratocytes.

Cell-crawling migration plays an essential role in complex biological phenomena. It is now generally believed that many processes essential to such migration are regulated by microtubules in many cells, including fibroblasts and neurons. However, keratocytes treated with nocodazole, which is an inhibitor of microtubule polymerization - and even keratocyte fragments that contain no microtubules - migrate at the same velocity and with the same directionality as normal keratocytes. In this study, we discovered that not only these migration properties, but also the molecular dynamics that regulate such properties, such as the retrograde flow rate of actin filaments, distributions of vinculin and myosin II, and traction forces, are also the same in nocodazole-treated keratocytes as those in untreated keratocytes. These results suggest that microtubules are not in fact required for crawling migration of keratocytes, either in terms of migrating properties or of intracellular molecular dynamics.

Structural requirements of strigolactones for germination induction and inhibition of Striga gesnerioides seeds.

KEY MESSAGE: Structure-activity relationship studies of strigolactones and Striga gesnerioides seed germination revealed strict structural requirements for germination induction and a new function of the plant hormones as germination inhibitors. Stereoisomers of the naturally occurring strigolactones, strigol, sorgolactone, orobanchol, sorgomol and 5-deoxystrigol, 36 in total, were prepared and screened for the ability to induce and/or inhibit the germination of Striga hermonthica and Striga gesnerioides seeds collected from mature plants that parasitized on sorghum and cowpea, respectively. All of the compounds induced S. hermonthica seed germination, albeit displayed differential activities. On the other hand, only a limited number of the compounds induced significant germination in S. gesnerioides, thus indicating strict structural requirements. Strigolactones inducing high germination in S. gesnerioides induced low germination in S. hermonthica. Strigolactones with the same configuration at C3a, C8b and C2' as that in 5-deoxystrigol (9a) induced high germination of S. hermonthica seeds, but most of them inhibited the germination of S. gesnerioides. The differential response of S. gesnerioides to strigolactones may play an important role in the survival of the species. However, the compounds could be used as means of control if mixed cropping of cowpea and sorghum is adopted.

The bioconversion of 5-deoxystrigol to sorgomol by the sorghum, Sorghum bicolor (L.) Moench.

Strigolactones, important rhizosphere signalling molecules and a class of phytohormones that control shoot architecture, are apocarotenoids of plant origin. They have a structural core consisting of a tricyclic lactone connected to a butyrolactone group via an enol ether bridge. Deuterium-labelled 5-deoxystrigol stereoisomers were administered to aquacultures of a high sorgomol-producing sorghum cultivar, Sorghum bicolor (L.) Moench, and conversion of these substrates to sorgomol stereoisomers was investigated. Liquid chromatography-mass spectrometry analyses established that 5-deoxystrigol (5-DS) and ent-2'-epi-5-deoxystrigol were absorbed by sorghum roots, converted to sorgomol and ent-2'-epi-sorgomol, respectively, and exuded out of the roots. The conversion was inhibited by uniconazole-P, implying the involvement of cytochrome P450 in the hydroxylation. These results provide experimental evidence for the postulated biogenetic scheme for formation of strigolactones, in which hydroxylation at C-9 of 5-DS can generate sorgomol.

Design of novel hypoxia-targeting IDO hybrid inhibitors conjugated with an unsubstituted L-TRP as an IDO affinity moiety.

We presented here design, syntheses and inhibitory activities of novel hypoxia-targeting IDO hybrid inhibitors conjugated with an unsubstituted L-Trp as an IDO affinity moiety without inhibitor 1MT, such as L-Trp-TPZ hybrids 1 (TX-2274), 2 (UTX-3), 3 (UTX-4), and 4 (UTX-2). TPZ-monoxide hybrids 1 and 3 were good competitive IDO inhibitors, while TPZ hybrids 2 and 4 were uncompetitive IDO inhibitors. Among them TPZ-monoxide hybrid 1 have the strongest IDO inhibitory activity. It suggests that TPZ-monoxide hybrids 1 and 3 are able to bind the active site of IDO, TPZ hybrids 2 and 4 are able to bind the enzyme-substrate complex. We proposed the possible mechanism of action of TPZ hybrid 2 that may first affect as a hypoxic cytotoxin, and then metabolized to TPZ-monoxide hybrid 1, which may do as an IDO inhibitor more effectively than its parent TPZ hybrid 2.

Synthesis and biological activity of 1-methyl-tryptophan-tirapazamine hybrids as hypoxia-targeting indoleamine 2,3-dioxygenase inhibitors.

We have designed and synthesized new hypoxic-neoplastic cells-targeted indoleamine 2,3-dioxygenase (IDO) inhibitors. 1-Methyl-tryptophan (1MT)-tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine 1,4-dioxide) hybrid inhibitors including 1 (TX-2236), 2 (TX-2235), 3 (TX-2228), and 4 (TX-2234) were prepared. All of these compounds were uncompetitive IDO inhibitors. TPZ-monoxide hybrids 1 and 3 showed higher IDO inhibitory activities than TPZ hybrids 2 and 4. Among these hybrids, hybrid 1 was the most potent IDO inhibitor. TPZ hybrids 2 and 4 showed stronger hypoxia-selective cytotoxicity than TPZ to EMT6/KU cells. These data suggest that TPZ hybrids 2 and 4 may act through their dual biological functions: first, they function as hypoxic cytotoxins in hypoxic cells, and then are metabolized to their TPZ-monoxide (3-amino-1,2,4-benzotriazine 1-oxide) hybrids, which function as IDO inhibitors.

Design of antiangiogenic hypoxic cell radiosensitizers: 2-nitroimidazoles containing a 2-aminomethylene-4-cyclopentene-1,3-dione moiety.

We designed chiral 2-nitroimidazole derivatives containing a 2-aminomethylene-4-cyclopentene-1,3-dione moiety as antiangiogenic hypoxic cell radiosensitizers. Based on results of molecular orbital calculations, the 2-aminomethylene-4-cyclopentene-1,3-dione moiety is expected to show high electrophilicity comparable to that of the 2-methylene-4-cyclopentene-1,3-dione moiety included in TX-1123 and tyrphostin AG17. We evaluated the antiangiogenic and radiosensitizing effects of the new compounds, along with other biological properties including their activities as hypoxic cytotoxicities and protein tyrosine kinase (PTK) inhibitory activities. Among the compounds tested, 5 (TX-2036) proved to be the strongest antiangiogenic hypoxic cell radiosensitizer. All the other chiral 2-nitroimidazole derivatives having 2-aminomethylene-4-cyclopentene-1,3-dione moiety tested were also antiangiogenic hypoxic cell radiosensitizers. The PTK inhibitory activity of 5 (TX-2036) showed this to be a promising and potent EGFR kinase inhibitor, having an IC(50) value of lower than 2microM. This compound also was an Flt-1 kinase inhibitor having an IC(50) value of lower than 20microM. Our results show that these chiral 2-nitroimidazole derivatives that contain the 2-aminomethylene-4-cyclopentene-1,3-dione moiety as a potent antiangiogenic pharmacophoric descriptor are promising lead candidates for the development of antiangiogenic hypoxic cell radiosensitizers.