Glycation promotes pulp calcification in Type 2 diabetes rat model.

OBJECTIVES: Intrapulpal calcifications can occur in the dental pulp of patients with diabetes. We focused on the association between ectopic calcifications in the dental pulp and advanced glycation end products (AGEs) in Spontaneously Diabetic Torii (SDT)-fatty rats, an obese type 2 diabetic rat model, to determine the mechanism of calcification with pulp stone in the dental pulp. MATERIALS AND METHODS: Pathologic calcification in the dental pulp of SDT-fatty rats was observed using electron microscopy and immunohistochemical analysis. Moreover, mechanical analysis of periapical region of molar tooth against occlusal force was performed. RESULTS: In SDT-fatty rats, pathogenic pulpal calcifications occurred during blood glucose elevation after 6 weeks, and granular calcification was observed in the dental pulp after 11 weeks. Pentosidine, a major AGE, and the receptor for AGEs were strongly expressed in the dental pulp of SDT-fatty rats. S100A8, TNF-α, and IL-6 also showed positive response in the dental pulp of the SDT-fatty rat, which indicated pulpal inflammation. Blood flow disorder and hypoxic dental pulp cells were also observed. In silico simulation, strain from occlusal force concentrates on the root apex. CONCLUSIONS: Glycation makes blood vessels fragile, and occlusal forces damage the vessels mechanically. These are factors for intrapulpal calcification of diabetes.

Inhibition of STAT-mediated cytokine responses to chemically-induced colitis prevents inflammation-associated neurobehavioral impairments.

Depression can be associated with chronic systemic inflammation, and production of peripheral proinflammatory cytokines and upregulation of the kynurenine pathway have been implicated in pathogenesis of depression. However, the mechanistic bases for these comorbidities are not yet well understood. As tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO), which convert tryptophan to kynurenine, are rate-limiting enzymes of the kynurenine pathway, we screened TDO or IDO inhibitors for effects on the production of proinflammatory cytokines in a mouse macrophage cell line. The TDO inhibitor 680C91 attenuated LPS-induced pro-inflammatory cytokines including IL-1ß and IL-6. Surprisingly, this effect was TDO-independent, as it occurred even in peritoneal macrophages from TDO knockout mice. Instead, the anti-inflammatory effects of 680C91 were mediated through the suppression of signal transducer and activator of transcription(STAT) signaling. Furthermore, 680C91 suppressed production of proinflammatory cytokines and STAT signaling in an animal model of inflammatory bowel disease. Specifically, 680C91 effectively attenuated acute phase colon cytokine responses in male mice subjected to dextran sulfate sodium (DSS)-induced colitis. Interestingly, this treatment also prevented the development of anxiodepressive-like neurobehaviors in DSS-treated mice during the recovery phase. The ability of 680C91 to prevent anxiodepressive-like behavior in response to chemically-induced colitis appeared to be due to rescue of attenuated dopamine responses in the nucleus accumbens. Thus, inhibition of STAT-mediated, but TDO-independent proinflammatory cytokines in macrophages can prevent inflammation-associated anxiety and depression. Identification of molecular mechanisms involved may facilitate the development of new treatments for gastrointestinal-neuropsychiatric comorbidity.

Subregion-Specific Regulation of Dopamine D1 Receptor Signaling in the Striatum: Implication for L-DOPA-Induced Dyskinesia.

The striatum is the main structure of the basal ganglia. The striatum receives inputs from various cortical areas, and its subregions play distinct roles in motor and emotional functions. Recently, striatal maps based on corticostriatal connectivity and striosome-matrix compartmentalization were developed, and we were able to subdivide the striatum into seven subregions. Dopaminergic modulation of the excitability of medium spiny neurons (MSNs) is critical for striatal function. In this study, we investigated the functional properties of dopamine signaling in seven subregions of the striatum from male mice. By monitoring the phosphorylation of PKA substrates including DARPP-32 in mouse striatal slices, we identified two subregions with low D1 receptor signaling: the dorsolateral portion of the intermediate/rostral part (DL-IR) and the intermediate/caudal part (IC). Low D1 receptor signaling in the two subregions was maintained by phosphodiesterase (PDE)10A and muscarinic M4 receptors. In an animal model of 6-hydroxydopamine (6-OHDA)-induced hemi-parkinsonism, D1 receptor signaling was upregulated in almost all subregions including the DL-IR, but not in the IC. When L-DOPA-induced dyskinesia (LID) was developed, D1 receptor signaling in the IC was upregulated and correlated with the severity of LID. Our results suggest that the function of the striatum is maintained through the subregion-specific regulation of dopamine D1 receptor signaling and that the aberrant activation of D1 receptor signaling in the IC is involved in LID. Future studies focusing on D1 receptor signaling in the IC of the striatum will facilitate the development of novel therapeutics for LID.SIGNIFICANCE STATEMENT Recent progress in striatal mapping based on corticostriatal connectivity and striosome-matrix compartmentalization allowed us to subdivide the striatum into seven subregions. Analyses of D1 receptor signaling in the seven subregions identified two unique subregions with low D1 receptor signaling: the dorsolateral portion of the intermediate/rostral part (DL-IR) and the intermediate/caudal part (IC). Aberrant activation of D1 receptor signaling in the IC is involved in L-DOPA-induced dyskinesia (LID). Previous studies of LID have mainly focused on the DL-IR, but not on the IC of the striatum. Future studies to clarify aberrant D1 receptor signaling in the IC are required to develop novel therapeutics for LID.

Effects of advanced glycation end products on dental pulp calcification.

OBJECTIVE: The main aim of this study was to elucidate the effects of advanced glycation end products (AGEs) on the calcification of cultured rat dental pulp cells (RDPCs) and to investigate the crystallisation ability of glycated collagen. MATERIALS AND METHODS: AGEs were prepared via non-enzymatic glycation of a dish coated with type I collagen using dl-glyceraldehyde. To investigate the effects of AGEs on RDPCs, we performed WST-1 and lactate dehydrogenase assays; alkaline phosphatase, Alizarin Red S and immunohistochemical staining; and real-time quantitative reverse transcription PCR. In addition, we performed crystallisation experiments on glycated collagen. All microstructures were analysed using scanning electron microscopy/energy-dispersive X-ray spectroscopy and transmission electron microscopy/diffraction pattern analysis. RESULTS: AGEs did not affect the proliferation or differentiation of RDPCs, but enhanced the calcification rate and cytotoxicity. No major calcification-related genes or proteins were involved in these calcifications, and glycated collagen was found to exhibit a negative polarity and form calcium phosphate crystals. Cytotoxicity due to drastic changes in the concentration of pericellular ions led to dystrophic calcification, assumed to represent an aspect of diabetic pulp calcifications. CONCLUSION: Glycated collagen-containing AGEs provide a nurturing environment for crystallisation and have a significant effect on the early calcification of RDPCs.

Varenicline aggravates plaque formation through α7 nicotinic acetylcholine receptors in ApoE KO mice.

Varenicline is one of the most widely used drugs for smoking cessation. However, whether an adverse effect of varenicline is associated with the risk of serious cardiovascular events remains controversial. In this study, we determined if varenicline increases the risk of cardiovascular events using apolipoprotein E knockout (ApoE KO) mice. ApoE KO mice (8 weeks old) were injected with varenicline 0.5 mg kg(-1)day(-1) for 3 weeks. Varenicline aggravated atherosclerotic plaque formation in whole aorta from ApoE KO mice compared with vehicle. Methyllycaconitine, an α7 nicotinic acetylcholine receptor (nAChR) antagonist, inhibited varenicline-induced aggravated plaque formation. Our findings show that varenicline progresses atherosclerotic plaque formation through α7 nAChR, and thereby increases the risk of cardiovascular events.

Direct synthesis of cyanate anion from dinitrogen catalysed by molybdenum complexes bearing pincer-type ligand.

Dinitrogen is an abundant and promising material for valuable organonitrogen compounds containing carbon-nitrogen bonds. Direct synthetic methods for preparing organonitrogen compounds from dinitrogen as a starting reagent under mild reaction conditions give insight into the sustainable production of valuable organonitrogen compounds with reduced fossil fuel consumption. Here we report the catalytic reaction for the formation of cyanate anion (NCO-) from dinitrogen under ambient reaction conditions. A molybdenum-carbamate complex bearing a pyridine-based 2,6-bis(di-tert-butylphosphinomethyl)pyridine (PNP)-pincer ligand is synthesized from the reaction of a molybdenum-nitride complex with phenyl chloroformate. The conversion between the molybdenum-carbamate complex and the molybdenum-nitride complex under ambient reaction conditions is achieved. The use of samarium diiodide (SmI2) as a reductant promotes the formation of NCO- from the molybdenum-carbamate complex as a key step. As a result, we demonstrate a synthetic cycle for NCO- from dinitrogen mediated by the molybdenum-PNP complexes in two steps. Based on this synthetic cycle, we achieve the catalytic synthesis of NCO- from dinitrogen under ambient reaction conditions.

Biological Evaluation of the Effect of Root Canal Sealers Using a Rat Model.

Gutta-percha points and root canal sealers have been used for decades in endodontics for root canal obturation. With techniques such as single cone methods, the amount of sealer is larger, making their properties more critical. However, relatively few reports have comprehensively evaluated their biological effects. To this end, we evaluated three types of sealers, zinc oxide-fatty acid-, bio-glass- and methacrylate resin-containing sealers were considered. Their biological effects were evaluated using a rat subcutaneous implantation model. Each sealer was loaded inside a Teflon tube and implanted subcutaneously in the backs of rats. Inflammatory cells were observed around all samples 7 days after implantation and reduced after 28 days. Our results revealed that all samples were in contact with the subcutaneous tissue surrounding the sealer. Additionally, Ca and P accumulation was observed in only the bio-glass-containing sealer. Furthermore, each of the three sealers exhibited unique immune and inflammatory modulatory effects. In particular, bio-glass and methacrylate resin sealers were found to induce variable gene expression in adjacent subcutaneous tissues related to angiogenesis, wound healing, muscle tissue, and surrounding subcutaneous tissue. These results may help to understand the biological impacts of root canal sealers on surrounding biological tissues, guiding future research and comparisons with new generations of materials.

Varenicline enhances oxidized LDL uptake by increasing expression of LOX-1 and CD36 scavenger receptors through α7 nAChR in macrophages.

Varenicline is a widely used and effective drug for smoking cessation. It is a partial agonist of the α4ß2 nicotinic acetylcholine receptor (nAChR) and full agonist of α7 nAChR. We have reported that varenicline aggravates formation of atherosclerotic plaques through α7 nAChR in apolipoprotein E knockout mice. However, little is known about its effects on macrophages in atherosclerotic plaques. Here, we ascertained whether varenicline promotes oxidized low-density lipoprotein (oxLDL) uptake in mouse peritoneal macrophages in vitro and clarified its mechanism. We investigated the effects of varenicline (1-10µM) on expression of scavenger receptors (lectin-like oxidized LDL receptor-1 (LOX-1), cluster of differentiation (CD) 36 and scavenger receptor class A (SR-A)) in RAW264.7 cells. Expression of protein and mRNA was determined by western blotting and real-time quantitative reverse transcription-polymerase chain reaction, respectively. Effects of varenicline (10µM) on oxLDL uptake were examined by counting the number of macrophages stained with oil red O and hematoxylin. Varenicline significantly increased expression of the protein and mRNA of LOX-1 and CD36, but not SR-A, in RAW264.7 cells, and increased oxLDL uptake in macrophages. These effects of varenicline were blocked significantly by an α7 nAChR antagonist, methyllycaconitine (MLA) (50nM), but not by an α4ß2 nAChR antagonist, dihydro-ß-erythroidine hydrobromide (DHßE) (1µM). These data suggest that varenicline promotes oxLDL uptake by upregulating expression of LOX-1 and CD36 through α7 nAChR in macrophages. We found that varenicline significantly activated extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor-kappa B (NF-κB) signaling pathways in RAW264.7 cells. This activation was blocked by MLA but not DHßE. Therefore, ERK1/2-NF-κB signaling pathway is highly likely to be responsible for varenicline-induced upregulation of LOX-1 and CD36 expression through α7 nAChR in macrophages. These processes probably contribute to varenicline-aggravated atherosclerotic plaque formation. Hence, an increased risk of cardiovascular events upon varenicline treatment could occur, and must be considered in patients (especially those suffering from cardiovascular diseases).

Varenicline promotes endothelial cell migration by lowering vascular endothelial-cadherin levels via the activated α7 nicotinic acetylcholine receptor-mitogen activated protein kinase axis.

Varenicline is a widely used and effective drug for smoking cessation. Despite its efficacy, varenicline increases the risk of cardiovascular disease. We previously demonstrated that varenicline aggravates atherosclerotic plaque formation in apolipoprotein E knockout mice. However, little is known about its effects in vascular endothelial cells. Therefore, we examined whether varenicline promotes migration of human umbilical vein endothelial cells (HUVECs) using the Boyden chamber assay. Varenicline (100µM) markedly promoted migration of HUVECs and decreased expression of vascular endothelial (VE)-cadherin, an endothelial adhesion molecule. Extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) signaling were markedly activated by varenicline. Methyllycaconitine (MLA; 100nM), an α7 nicotinic acetylcholine receptor (nAChR) antagonist, but not dihydro-ß-erythroidine hydrobromide (DHßE; 20µM) blocked varenicline-stimulated migration and varenicline-activated ERK, p38 and JNK signaling in HUVECs. MLA (100nM), PD98059 (an ERK inhibitor; 20µM), SB203580 (a p38 inhibitor; 20µM) and SP600125 (a JNK inhibitor; 20µM) also blocked cell migration and varenicline-induced downregulation of VE-cadherin expression in HUVECs. These findings suggest that varenicline promotes HUVEC migration by lowering VE-cadherin expression due to activated ERK/p38/JNK signaling through α7 nAChR. These processes probably contribute to varenicline-aggravated atherosclerotic plaque. Hence, an increased risk of cardiovascular events upon varenicline treatment might occur and must be considered in patients with cardiovascular diseases.