Cyclic guanosine monophosphate-adenosine monophosphate synthetase/stimulator of interferon genes signaling aggravated corneal allograft rejection through neutrophil extracellular traps.

The activation of innate immunity following transplantation has been identified as a crucial factor in allograft inflammation and rejection. However, the role of cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)/stimulator of interferon genes (STING) signaling-mediated innate immunity in the pathogenesis of allograft rejection remains unclear. Utilizing a well-established murine model of corneal transplantation, we demonstrated increased expression of cGAS and STING in rejected-corneal allografts compared with syngeneic (Syn) and normal (Nor) corneas, along with significant activation of the cGAS/STING pathway, as evidenced by the enhanced phosphorylation of TANK-binding kinase 1and interferon regulatory factor 3. Pharmacological and genetic inhibition of cGAS/STING signaling markedly delayed corneal transplantation rejection, resulting in prolonged survival time and reduced inflammatory infiltration. Furthermore, we observed an increase in the formation of neutrophil extracellular traps (NETs) in rejected allografts, and the inhibition of NET formation through targeting peptidylarginine deiminase 4 and DNase I treatment significantly alleviated immune rejection and reduced cGAS/STING signaling activity. Conversely, subconjunctival injection of NETs accelerated corneal transplantation rejection and enhanced the activation of the cGAS/STING pathway. Collectively, these findings demonstrate that NETs contribute to the exacerbation of allograft rejection via cGAS/STING signaling, highlighting the targeting of the NETs/cGAS/STING signaling pathway as a potential strategy for prolonging allograft survival.

Exosomal miR-181d-5p Derived from Rapamycin-Conditioned MDSC Alleviated Allograft Rejection by Targeting KLF6.

Immune rejection and side effects of long-term administration of immunosuppressants are the two major obstacles to allograft acceptance and tolerance. The immunosuppressive extracellular vesicles (EVs)-based approach has been proven to be effective in treating autoimmune/inflammatory disorders. Herein, the anti-rejection advantage of exosomes (Rapa-Exo) from rapamycin-conditioned myeloid-derived suppressor cells (MDSCs) over exosomes (Exo-Nor) from the untreated MDSCs is shown. The exosomal small RNA sequencing and loss-of-function assays reveal that the anti-rejection effect of Rapa-Exo functionally relies on miR-181d-5p. Through target prediction and double-luciferase reporter assay, Kruppel-like factor (KLF) 6 is identified as a direct target of miR-181d-5p. Finally, KLF6 knockdown markedly resolves inflammation and prolongs the survival of corneal allografts. Taken together, these findings support that Rapa-Exo executes an anti-rejection effect, highlighting the immunosuppressive EVs-based treatment as a promising approach in organ transplantation.

Microbiological Characteristics and Risk Factors Involved in Progression from Fungal Keratitis with Hypopyon to Keratitis-Related Endophthalmitis.

PURPOSE: To investigate the differences in microbiological characteristics, risk factors, drug resistance, and visual outcomes in three infections: fungal keratitis with hypopyon (FKH), keratitis-related fungal endophthalmitis (FKE), and fungal endophthalmitis without keratitis (FE). METHODS: An analytical cross-sectional study. RESULTS: In total, 14.57% of eyes with FKH progressed to endophthalmitis. Hypopyon, pre-existence of lens problems, topical steroid use and sever keratitis were significantly associated with the development of FKE. The risk factors of the FKH and FE group were mainly plant trauma and open globe trauma, respectively. Keratitis-related endophthalmitis (FKE) showed a significantly higher resistance than the other two groups. The FKH group had the best final visual acuity, while the FKE group had the worst. CONCLUSION: Hypopyon height, pre-existing lens problems, topical steroid use and sever keratitis are risk factors for progression to endophthalmitis in eyes with fungal keratitis, and its progression is not affected by a single fungus. The antifungal drugs resistance in patients with endophthalmitis related to keratitis was significantly higher than that associated with other reasons. Timely diagnosis and risk factor assessment are essential for ensuring early treatment of FKE.

The short-term effects of blood donation on the ocular parameters including blood flow of the retina and choroid in healthy people using OCT- angiography.

BACKGROUND: To investigate the short-term effects of blood donation on the morphology and blood flow of the retina and choroid in healthy people using optical coherence tomography angiography (OCTA). METHODS: Twenty-eight healthy blood donors (56 eyes) who participated in the 200 ml voluntary blood donation between March 2, 2021 and January 20, 2022 were included. The best corrected visual acuity (BCVA), systolic (SBP) and diastolic blood pressure (DBP), intraocular pressure (IOP), subfoveal choroid thickness (SFCT), retinal thickness (RT), retinal superficial vascular density (SVD), deep vascular density (DVD) and foveal avascular were a (FAZ) were measured and statistically analysed 10 min before, 30 min and 24 h after the blood donation. RESULTS: The 200 ml blood donation could cause significant IOP reduction at 24 h (P = 0.006), which was negatively correlated with SBP (r = -0.268, P = 0.046), while SBP, DBP, or ocular perfusion pressure were not affected (> 0.05). Moreover, no significant difference existed in the OCT and OCTA indexes, including SFCT, RT, SVD, DVD, and FAZ, before and after the 200 ml blood donation (P > 0.05). The visual acuity was not affected either (P > 0.05). CONCLUSIONS: The 200 ml blood donation was noted to be associated with statistically significant IOP reduction at 24 h, while SBP, DBP, or OPP was not affected. The blood flow of the retina and choroid or the visual acuity did not change significantly after the blood donation. Larger studies with different volumes of blood donation were needed to further analysis the effect of blood donation on ocular parameters.

Risk factors and microbiological characteristics: from bacterial keratitis with hypopyon to keratitis-related endophthalmitis.

PURPOSE: To compare the clinical and microbiological characteristics in patients with bacterial keratitis with hypopyon (BKH), bacterial keratitis-related endophthalmitis (BKE), and bacterial endophthalmitis without keratitis (BE). METHODS: Data from all inpatients who were clinically diagnosed with BKH, BKE, and BE from 2018 to 2020 were collected retrospectively. The demographics, predisposing risk factors, clinical characteristics, microbiological profiles, and antibiotic susceptibility of the patients were evaluated. RESULTS: Approximately 9.46% (28/296) of eyes with BKH progressed to endophthalmitis. The hypopyon (OR = 5.35, 95% CI: 2.17-7.08) and corneal perforation (OR = 2.47, 95% CI: 1.04-4.86) were significantly related to the development of BKE. The odds ratios for hypopyon of less than 1 mm, 1-3 mm, and greater than 3 mm were 1, 2.09 (95% CI: 1.17-3.15), and 4.12 (95% CI:2.59-5.68), respectively. The predominant causative pathogen was Staphylococcus epidermidis (36.43%, 38.89%), followed by Streptococci (14.73%, 16.67%), Staphylococcus aureus (8.53%, 7.79%), and Pseudomonas aeruginosa (9.30%, 7.14%) in eyes with BKH and BE. However, the main pathogens were Pseudomonas aeruginosa (37.50%) and Staphylococcus aureus (31.25%) in eyes with BKE. In the BKH, BKE, and BE groups, almost 100% of Staphylococcus aureus isolates were sensitive to vancomycin (97.70%, 100%, 95.56%), about a half were sensitive to fluoroquinolones (51.85%, 39.90%, 62.34%), and approximately 30% were sensitive to trimethoprim/sulfa (27.77%, 21.56%, 33.56%) and cefazolin (41.47%, 20.31%, 38.81%). The susceptibility of Pseudomonas aeruginosa to fluoroquinolones antibiotics was 55.75%, 66.67%, and 62.58%, respectively, in the three groups. CONCLUSIONS: The height of hypopyon and corneal perforation are risk factors for progression to endophthalmitis in eyes with bacterial keratitis. When Staphylococcus aureus and Pseudomonas aeruginosa are identified, vigilance is required for advanced endophthalmitis.

Synthesis and Evaluation of [11C]7-Halogen-2-Phenyl Isoindolone Derivatives: Potential PET Radioligands for in vivo Imaging of 5-HT2 C Receptors.

The serotonin 5-HT2 C receptor (5-HT2 C R) is abundantly expressed throughout the central nervous system, and involved in a variety of neuroendocrine and neurobehavioral processes. The development of a selective radioligand that will enable in vivo imaging and quantification of 5-HT2 C R densities represents a significant technological advancement in understanding both the normal function and pathophysiology of the 5-HT2 C R. Four 7-halogen-2-phenyl isoindolones (7-F, Cl, Br, I) were synthesized and displayed high affinities for 5-HT2 C R and high selectivity over 5-HT2 A and 5-HT2 B . [11C]7-Chloro-2-[4-methoxy-3-[2-(4-methylpiperidin-1-yl)ethoxy]phenyl]isoindolin-1-one (6) and [11C]7-iodo-2-[4-methoxy-3-[2-(4-methylpiperidin-1-yl)ethoxy]phenyl]isoindolin-1-one (9) were synthesized in high radiochemical yield of 37-44% [n = 10, decay corrected from end of (11C)CH3I synthesis] with high radiochemical purity via O-methylation with [11C]CH3I, respectively. MicroPET imaging studies in male rats with or without 5-HT2 C antagonist SB-242084 showed that [11C]6 and [11C]9 display specific bindings to 5-HT2 C R in the choroid plexus and hippocampus. In vivo microPET brain imaging studies in rhesus monkeys demonstrated that [11C]6 and [11C]9 exhibit excellent blood-brain barrier penetration. The contrast of bindings to the choroid plexus and hippocampus compared to the cerebellum peaked at 2.7 and 1.6, respectively, for [11C]6, and 3.7 and 2.7, respectively, for [11C]9, which were reduced by administration of a dose of SB-242084. Our results support the candidacy of [11C]6 and [11C]9 for further study as radioligands for in vivo quantitation of 5-HT2 C sites by PET.

Macrophage migration inhibitory factor drives pathology in a mouse model of spondyloarthritis and is associated with human disease.

Spondyloarthritis (SpA), a type 3 immunity-mediated inflammatory arthritis, is a systemic rheumatic disease that primarily affects the joints, spine, gut, skin, and eyes. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine, yet MIF's pathological role in SpA is unknown. Here, we observed that the expression of MIF and its receptor CD74 is increased in blood and tissues of curdlan (ß-glucan)­treated SKG mice, a mouse model of SpA. We found that neutrophils substantially expanded and produced MIF in curdlan-treated SKG mice and that human neutrophils from SpA patients secreted higher concentrations of MIF compared to healthy individuals. Although genetic deletion of Mif (Mif−/−) substantially suppressed the severity of SpA features, adoptive transfer of inflammatory neutrophils induced SpA pathology in curdlan-treated Mif−/− SKG mice; in contrast, blocking the function of neutrophils with anti­Gr-1 antibody suppressed the curdlan-induced SpA-like phenotype. We also determined that systemic MIF overexpression was sufficient to induce SpA-like clinical features in SKG mice with enhanced type 3 immunity, whereas SKG mice treated with a MIF antagonist prevented or attenuated curdlan-induced SpA manifestations. Mechanistically, we identified that MIF intensifies type 3 immunity by boosting human and mouse T regulatory cell (Treg) acquisition of a TH17 cell­like phenotype, including the up-regulation of interleukin-17 (IL-17) and IL-22 in vitro. Tregs in blood and synovial fluids from SpA patients have a pathologic TH17 phenotype. These results indicate that MIF is a crucial regulator and a potential therapeutic target in type 3 immunity-mediated arthritis.

Prenatal exercise reprograms the development of hypertension progress and improves vascular health in SHR offspring.

BACKGROUND: Upregulation of L-type voltage-gated Ca2+ (CaV1.2) channel in the arterial myocytes is a hallmark feature of hypertension. However, whether maternal exercise during pregnancy has a sustained beneficial effect on the offspring of spontaneously hypertensive rats (SHRs) through epigenetic regulation of CaV1.2 channel is largely unknown. METHODS: Pregnant SHRs and Wistar-Kyoto rats were subjected to swimming and the vascular molecular and functional properties of male offspring were evaluated at embryonic (E) 20.5 day, 3 months (3 M), and 6 months (6 M). RESULTS: Exercise during pregnancy significantly decreased the resting blood pressure at 3 M but not 6 M in the offspring of SHR. Prenatal exercise significantly reduced the cardiovascular reactivity, the contribution of CaV1.2 channel to the vascular tone, and the whole-cell current density of CaV1.2 channel in both 3 M and 6 M offspring of SHR. Moreover, maternal exercise triggered hypermethylation of the promoter region of the CaV1.2 α1C gene (CACNA1C), with a concomitant decrease in its protein and mRNA expressions in SHR offspring at E20.5, 3 M, and 6 M. Tissue culture experiments further confirmed that 5-Aza-2'-deoxycytidine increased the structure and functional expression of CaV1.2 channel by inhibiting the DNA methylation of CACNA1C. However, the improvement of prenatal exercise on the blood pressure, function, and expression of CaV1.2 channel was attenuated in the offspring of SHRs at 6 M compared to the 3 M readout. CONCLUSIONS: These data suggest that prenatal exercise improves the vascular function by the hypermethylation of CACNA1C in the arterial myocytes and delays the development of hypertension in the offspring of SHRs. However, these effects fade out with age.

Comparison of the Efficacy and Safety of Intravitreal Conbercept with Intravitreal Ranibizumab for Treatment of Diabetic Macular Edema: A Meta-Analysis.

METHODS: Relevant studies were identified through systemic searches of PubMed, Embase, Cochrane Library, Ovid, CNKI, and Wanfang database up to 28 February 2019. Changes in central retinal thickness (CRT) in µm and best-corrected visual acuity (BCVA) in logMAR equivalents at 1, 3, and 6 months after initial treatment were performed by pooled analysis. Adverse events (AEs) were evaluated. RESULTS: Eight articles involving 588 patients with DME were identified for this meta-analysis. The results showed that IVC significantly improved BCVA compared with IVR at 6 mo (SMD = -0.74 95% CI: -1.28 to -0.2; p=0.029) in patients with DME. IVC was superior to IVR in reducing central retinal thickness (CRT at 1 mo (p < 0.0001), 3 mo (p=0.025), and 6 mo (p=0.019)) from baseline with statistical significance. For AEs, the pooled results showed that no significant difference in the risk of intraocular pressure increased (OR = 1.71; 95% CI: 0.55 to 5.25; p=0.352) or conjunctival hemorrhage (OR = 0.89; 95% CI: 0.34 to 2.34; p=0.65) between two groups. CONCLUSIONS: This meta-analysis showed that IVC trended to be more effective than IVR in terms of functional and anatomic outcomes for treating DME.

Role of the Balance of Akt and MAPK Pathways in the Exercise-Regulated Phenotype Switching in Spontaneously Hypertensive Rats.

The mechanisms regulating vascular smooth muscle cell (VSMC) phenotype switching and the critical signal modulation affecting the VSMCs remain controversial. Physical exercise acts as an effective drug in preventing elevated blood pressure and improving vascular function. This study was designed to explore the influence of aerobic exercise on the suppression of VSMC phenotype switching by balancing of the Akt, also known as PKB (protein kinase B) and mitogen-activated protein kinase (MAPK) signaling pathways. Spontaneously hypertensive rats (SHRs) and normotensive rats were subjected to exercise treatment before measuring the vascular morphological and structural performances. Exercise induced reverse expression of VSMC protein markers (α-SM-actin, calponin, and osteopontin (OPN)) in spontaneously hypertensive rats. It is noteworthy that the low expression of phosphorylated Akt significantly decreased the expression of VSMC contractile phenotype markers (α-SM-actin and calponin) and increased the expression of the VSMC synthetic phenotype marker (OPN). However, the MAPK signal pathway exerts an opposite effect. VSMCs and whole vessels were treated by inhibitors, namely the p-Akt inhibitor, p-ERK inhibitor, and p-p38 MAPK inhibitors. VSMC phenotype markers were reversed. It is important to note that a significant reverse regulatory relationship was observed between the expression levels of MAPK and the contractile markers in both normotensive and spontaneously hypertensive rats. We demonstrate that aerobic exercise regulates the VSMC phenotype switching by balancing the Akt and MAPK signaling pathways in SHRs.

Exercise during pregnancy enhances vascular function via epigenetic repression of CaV1.2 channel in offspring of hypertensive rats.

AIMS: Studies suggest that cardiovascular function in offspring can be epigenetically programmed by environmental changes during pregnancy. CaV1.2 channel plays a major role in the regulation of the vascular tone. This study investigated the effects and underlying mechanisms of exercise during pregnancy on CaV1.2 channel functional remodeling in hypertensive offspring. MAIN METHODS: Exercise groups were subjected to swimming at the first day of pregnancy and on a regular schedule thereafter for 3 weeks. Their offspring (6-month-old, male) were tested for baseline blood pressure, cardiovascular response, and vascular tone of the mesenteric artery. Mesenteric artery smooth muscle cells were taken to study the whole-cell current of the CaV1.2 channel. Western blotting, RT-PCR and DNA bisulfite sequencing PCR were performed to study the protein, mRNA expression and DNA methylation of the CaV1.2 channel α1C subunit. KEY FINDINGS: Exercise during pregnancy reduced the pressor response to norepinephrine and Bay K8644, and the depressor response to nifedipine in offspring of hypertensive rats. The level of the CaV1.2 channel in norepinephrine-induced vasoconstrictions decreased, and the whole-cell current of the CaV1.2 channel declined in the SHR-EX group. Further studies found that exercise during pregnancy reduced the protein and mRNA expression of the CaV1.2 channel α1C subunit and upregulated DNA methylation of the Cacna1c gene promoter region in the hypertensive offspring. SIGNIFICANCE: These data suggest that exercise during pregnancy improves vascular functional remodeling in offspring of hypertensive rats, downregulating the CaV1.2 channel function and protein expression, a change that is most likely caused by DNA methylation.

The Na,K-ATPase in vascular smooth muscle cells.

The Na,K-ATPase is an enzyme essential for ion homeostasis in all cells. Over the last decades, it has been well-established that in addition to the transport of Na+/K+ over the cell membrane, the Na,K-ATPase acts as a receptor transducing humoral signals intracellularly. It has been suggested that ouabain-like compounds serve as endogenous modulators of this Na,K-ATPase signal transduction. The molecular mechanisms underlying Na,K-ATPase signaling are complicated and suggest the confluence of divergent biological pathways. This review discusses recent updates on the Na,K-ATPase signaling pathways characterized or suggested in vascular smooth muscle cells. The conventional view on this signaling is based on a microdomain structure where the Na,K-ATPase controls the Na,Ca-exchanger activity via modulation of intracellular Na+ in the spatially restricted submembrane space. This, in turn, affects intracellular Ca2+ and Ca2+ load in the sarcoplasmic reticulum leading to modulation of contractility as well as gene expression. An ion-transport-independent signal transduction from the Na,K-ATPase is based on molecular interactions. This was primarily characterized in other cell types but recently also demonstrated in vascular smooth muscles. The downstream signaling from the Na,K-ATPase includes Src and phosphatidylinositol-4,5-bisphosphate 3 kinase signaling pathways and generation of reactive oxygen species. Moreover, in vascular smooth muscle cells the interaction between the Na,K-ATPase and proteins responsible for Ca2+ homeostasis, e.g., phospholipase C and inositol triphosphate receptors, contributes to an integration of the signaling pathways. Recent update on the Na,K-ATPase dependent intracellular signaling and the significance for physiological functions and pathophysiological changes are discussed in this review.

microRNA-181a-5p antisense oligonucleotides attenuate osteoarthritis in facet and knee joints.

OBJECTIVES: We recently identified microRNA-181a-5p (miR-181a-5p) as a critical mediator involved in the destruction of lumbar facet joint (FJ) cartilage. In this study, we tested if locked nucleic acid (LNA) miR-181a-5p antisense oligonucleotides (ASO) could be used as a therapeutic to limit articular cartilage degeneration. METHODS: We used a variety of experimental models consisting of both human samples and animal models of FJ and knee osteoarthritis (OA) to test the effects of LNA-miR-181a-5p ASO on articular cartilage degeneration. Histopathological analysis including immunohistochemistry and in situ hybridisation were used to detect key OA catabolic markers and microRNA, respectively. Apoptotic/cell death markers were evaluated by flow cytometry. qPCR and immunoblotting were applied to quantify gene and protein expression. RESULTS: miR-181a-5p expression was increased in human FJ OA and knee OA cartilage as well as injury-induced FJ OA (rat) and trauma-induced knee OA (mouse) cartilage compared with control cartilage, correlating with classical OA catabolic markers in human, rat and mouse cartilage. We demonstrated that LNA-miR-181a-5p ASO in rat and mouse chondrocytes reduced the expression of cartilage catabolic and chondrocyte apoptotic/cell death markers in vitro. Treatment of OA-induced rat FJ or mouse knee joints with intra-articular injections of in vivo grade LNA-miR-181a-5p ASO attenuated cartilage destruction, and the expression of catabolic, hypertrophic, apoptotic/cell death and type II collagen breakdown markers. Finally, treatment of LNA-miR-181a-5p ASO in cultures of human knee OA chondrocytes (in vitro) and cartilage explants (ex vivo) further demonstrated its cartilage protective effects. CONCLUSIONS: Our data demonstrate, for the first time, that LNA-miR-181a-5p ASO exhibit cartilage-protective effects in FJ and knee OA.

Akt/eNOS and MAPK signaling pathways mediated the phenotypic switching of thoracic aorta vascular smooth muscle cells in aging/hypertensive rats.

Considerable evidence demonstrates that phenotypic switching of vascular smooth muscle cells (VSMCs) is influenced by aging and hypertension. During phenotypic switching, VSMCs undergo a switch to a proliferative and migratory phenotype, with this switch being a common pathology in cardiovascular diseases. The aim of this study was to explore the joint influence of age and hypertension on thoracic aortic smooth muscle phenotypic switching and the balance of Akt and mitogen-activated protein kinase (MAPK) signaling during this switch. Different ages of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were used to establish hypertension and aging models. The phenotypic state was determined by detecting the marker proteins alpha-SM-actin, calponin, and osteopontin (OPN) via immunohistochemical staining and Western blot. Signaling proteins associated with the Akt and MAPK pathways were detected in rat thoracic aorta using Western blot. Both aging and hypertension caused a decrease in contractile (differentiated) phenotype markers (alpha-SM-actin and calponin), while the synthetic (proliferative or de-differentiated) phenotype maker was elevated (OPN). When combining hypertension and aging, this effect was enhanced, with Akt signaling decreased, while MAPK signaling was increased. These results suggested that VSMCs phenotype switching is modulated by a balance between Akt and MAPK signaling in the process of aging and hypertension.

Akt modulation by miR-145 during exercise-induced VSMC phenotypic switching in hypertension.

AIMS: This study investigated whether long-term exercise can influence vascular smooth muscle cells (VSMCs) phenotypic switching in mesenteric arteries of hypertensive rats, with a focus on the modulation of protein kinase B (PKB/Akt) signaling by microRNA-145 (miR-145). MAIN METHODS: In the exercise intervention experiment, mesenteric arteries from 3-month-old spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) were isolated for histological observation, phenotypic marker analysis, Akt phosphorylation quantification, and miR-145 evaluation after being subjected to moderate-intensity treadmill training (E) or being sedentary (C) for 8 weeks. In the transfection experiment, VSMCs were harvested to determine Akt phosphorylation and mRNA expressions of the upstream and downstream signaling molecules. KEY FINDINGS: Calponin, a VSMC contractile marker, was significantly up-regulated in SHR-E relative to SHR-C (P < 0.05); while osteopontin (OPN), a dedifferentiation marker, was down-regulated in SHR-E relative to SHR-C (P < 0.05). Exercise significantly normalized the expression of miR-145 and significantly enhanced Akt phosphorylation (P < 0.05). In VSMCs over-expressing miR-145, Akt phosphorylation was significantly decreased (P < 0.05) with inhibited mRNA of both insulin-like growth factor 1 receptor (IGF-1R) and insulin receptor substrate 1 (IRS-1). In VSMCs transfected with miR-145 inhibitor, Akt phosphorylation and mRNA of IGF-1R and IRS-1 were all down-regulated. miR-145 did not exhibit a clear effect on p70 ribosomal kinase (p70S6K), the downstream of Akt, following the transfections. SIGNIFICANCE: Overall, exercise remodels arterioles in hypertension and induces VSMCs maintaining contractile phenotype, in which miR-145 appears to be involved by inversely regulating Akt signaling via its upstream signals.

Synthesis and Evaluation of Pyridyloxypyridyl Indole Carboxamides as Potential PET Imaging Agents for 5-HT2C Receptors.

Nine pyridyloxypyridyl indole carboxamides were synthesized and displayed high affinities for 5-HT2C receptors and high selectivity over 5-HT2A and 5-HT2B. Among them, 6-methyl-N-[6-[(2-methyl-3-pyridinyl)oxy]-3-pyridinyl]1H-indole-3-carboxamide (8) exhibits the highest 5-HT2C binding affinity (Ki = 1.3 nM) and high selectivity over 5-HT2A (∼1000 times) and 5-HT2B (∼140 times). [11C]8 was synthesized by palladium-catalyzed coupling reaction between pinacolboranate 16 and [11C]CH3I with an average radiochemical yield of 27 ± 4% (n = 8, decay-corrected from end of [11C]CH3I synthesis). MicroPET imaging studies in rhesus monkeys showed regional uptake of [11C]8 in the choroid plexus, whereas the bindings in all other brain regions were low. The specific binding in the choroid plexus was confirmed by administration of a blocking dose of 0.1 mg/kg of the 5-HT2C antagonist SB-242084.

Structural and Kinetic Studies of the Potent Inhibition of Metallo-ß-lactamases by 6-Phosphonomethylpyridine-2-carboxylates.

There are currently no clinically available inhibitors of metallo-ß-lactamases (MBLs), enzymes that hydrolyze ß-lactam antibiotics and confer resistance to Gram-negative bacteria. Here we present 6-phosphonomethylpyridine-2-carboxylates (PMPCs) as potent inhibitors of subclass B1 (IMP-1, VIM-2, and NDM-1) and B3 (L1) MBLs. Inhibition followed a competitive, slow-binding model without an isomerization step (IC50 values of 0.3-7.2 µM; Ki values of 0.03-1.5 µM). Minimum inhibitory concentration assays demonstrated potentiation of ß-lactam (Meropenem) activity against MBL-producing bacteria, including clinical isolates, at concentrations at which eukaryotic cells remain viable. Crystal structures revealed unprecedented modes of binding of inhibitor to B1 (IMP-1) and B3 (L1) MBLs. In IMP-1, binding does not replace the nucleophilic hydroxide, and the PMPC carboxylate and pyridine nitrogen interact closely (2.3 and 2.7 Å, respectively) with the Zn2 ion of the binuclear metal site. The phosphonate group makes limited interactions but is 2.6 Å from the nucleophilic hydroxide. Furthermore, the presence of a water molecule interacting with the PMPC phosphonate and pyridine N-C2 π-bond, as well as the nucleophilic hydroxide, suggests that the PMPC binds to the MBL active site as its hydrate. Binding is markedly different in L1, with the phosphonate displacing both Zn2, forming a monozinc enzyme, and the nucleophilic hydroxide, while also making multiple interactions with the protein main chain and Zn1. The carboxylate and pyridine nitrogen interact with Ser221 and -223, respectively (3 Å distance). The potency, low toxicity, cellular activity, and amenability to further modification of PMPCs indicate these and similar phosphonate compounds can be further considered for future MBL inhibitor development.

Resveratrol modulates cocaine-induced inhibitory synaptic plasticity in VTA dopamine neurons by inhibiting phosphodiesterases (PDEs).

Resveratrol is a natural phytoalexin synthesized by plants, including grapes. It displays a wide range of neuroprotective benefits associated with anti-aging. Recent studies have shown that resveratrol regulates dopaminergic transmission and behavioral effects of drugs of abuse. The goal of the present study is to investigate whether and how resveratrol alters basal inhibitory synaptic transmission and cocaine-induced inhibitory synaptic plasticity in dopamine neurons of the ventral tegmental area (VTA). We report that resveratrol elevated cAMP levels by itself and further potentiated a forskolin-induced increase in cAMP levels in midbrain slices, consistent with reported effects of inhibition of phosphodiesterases (PDEs). Resveratrol potentiated GABAA and GABAB-mediated inhibitory postsynaptic currents (IPSCs) in VTA dopamine neurons, and these effects were mediated by a protein kinase A (PKA)-dependent enhancement of presynaptic GABA release. In addition, we found that resveratrol blocked endocannabinoid-mediated long-term synaptic depression in VTA dopamine neurons. Resveratrol pretreatments attenuated cocaine-induced conditioned place preference and blocked the cocaine-induced reduction of GABAergic inhibition in VTA dopamine neurons. Together, these results provide evidence that resveratrol modulates basal inhibitory synaptic transmission, cocaine-induced synaptic plasticity, and drug-cue associative learning.

Macrophage Migration Inhibitory Factor Induces Inflammation and Predicts Spinal Progression in Ankylosing Spondylitis.

OBJECTIVE: To investigate the role of macrophage migration inhibitory factor (MIF) in the pathogenesis of ankylosing spondylitis (AS). METHODS: Patients who met the modified New York criteria for AS were recruited for the study. Healthy volunteers, rheumatoid arthritis patients, and osteoarthritis patients were included as controls. Based on the annual rate of increase in modified Stoke AS Spine Score (mSASSS), AS patients were classified as progressors or nonprogressors. MIF levels in serum and synovial fluid were quantitated by enzyme-linked immunosorbent assay. Predictors of AS progression were evaluated using logistic regression analysis. Immunohistochemical analysis of ileal tissue was performed to identify MIF-producing cells. Flow cytometry was used to identify MIF-producing subsets, expression patterns of the MIF receptor (CD74), and MIF-induced tumor necrosis factor (TNF) production in the peripheral blood. MIF-induced mineralization of osteoblast cells (SaOS-2) was analyzed by alizarin red S staining, and Western blotting was used to quantify active ß-catenin levels. RESULTS: Baseline serum MIF levels were significantly elevated in AS patients compared to healthy controls and were found to independently predict AS progression. MIF levels were higher in the synovial fluid of AS patients, and MIF-producing macrophages and Paneth cells were enriched in their gut. MIF induced TNF production in monocytes, activated ß-catenin in osteoblasts, and promoted the mineralization of osteoblasts. CONCLUSION: Our findings indicate an unexplored pathogenic role of MIF in AS and a link between inflammation and new bone formation.

Epigenetic regulation of L-type voltage-gated Ca2+ channels in mesenteric arteries of aging hypertensive rats.

Accumulating evidence has shown that epigenetic regulation is involved in hypertension and aging. L-type voltage-gated Ca2+ channels (LTCCs), the dominant channels in vascular myocytes, greatly contribute to arteriole contraction and blood pressure (BP) control. We investigated the dynamic changes and epigenetic regulation of LTCC in the mesenteric arteries of aging hypertensive rats. LTCC function was evaluated by using microvascular rings and whole-cell patch-clamp in the mesenteric arteries of male Wistar-Kyoto rats and spontaneously hypertensive rats at established hypertension (3 month old) and an aging stage (16 month old), respectively. The expression of the LTCC α1C subunit was determined in the rat mesenteric microcirculation. The expression of miR-328, which targets α1C mRNA, and the DNA methylation status at the promoter region of the α1C gene (CACNA1C) were also determined. In vitro experiments were performed to assess α1C expression after transfection of the miR-328 mimic into cultured vascular smooth muscle cells (VSMCs). The results showed that hypertension superimposed with aging aggravated BP and vascular remodeling. Both LTCC function and expression were significantly increased in hypertensive arteries and downregulated with aging. miR-328 expression was inhibited in hypertension, but increased with aging. There was no significant difference in the mean DNA methylation of CACNA1C among groups, whereas methylation was enhanced in the hypertensive group at specific sites on a CpG island located upstream of the gene promoter. Overexpression of miR-328 inhibited the α1C level of cultured VSMCs within 48 h. The results of the present study indicate that the dysfunction of LTCCs may exert an epigenetic influence at both pre- and post-transcriptional levels during hypertension pathogenesis and aging progression. miR-328 negatively regulated LTCC expression in both aging and hypertension.