GPCR Pharmacological Profiling of Aaptamine from the Philippine Sponge Stylissa sp. Extends Its Therapeutic Potential for Noncommunicable Diseases.

We report the first isolation of the alkaloid aaptamine from the Philippine marine sponge Stylissa sp. Aaptamine possessed weak antiproliferative activity against HCT116 colon cancer cells and inhibited the proteasome in vitro at 50 µM. These activities may be functionally linked. Due to its known, more potent activity on certain G-protein coupled receptors (GPCRs), including α-adrenergic and δ-opioid receptors, the compound was profiled more broadly at sub-growth inhibitory concentrations against a panel of 168 GPCRs to potentially reveal additional targets and therapeutic opportunities. GPCRs represent the largest class of drug targets. The primary screen at 20 µM using the ß-arrestin functional assay identified the antagonist, agonist, and potentiators of agonist activity of aaptamine. Dose-response analysis validated the α-adrenoreceptor antagonist activity of aaptamine (ADRA2C, IC50 11.9 µM) and revealed the even more potent antagonism of the ß-adrenoreceptor (ADRB2, IC50 0.20 µM) and dopamine receptor D4 (DRD4, IC50 6.9 µM). Additionally, aaptamine showed agonist activity on selected chemokine receptors, by itself (CXCR7, EC50 6.2 µM; CCR1, EC50 11.8 µM) or as a potentiator of agonist activity (CXCR3, EC50 31.8 µM; CCR3, EC50 16.2 µM). These GPCRs play a critical role in the treatment of cardiovascular disease, diabetes, cancer, and neurological disorders. The results of this study may thus provide novel preventive and therapeutic strategies for noncommunicable diseases (NCDs).

Modulation of the chemokine/chemokine receptor axis as a novel approach for glioma therapy.

Chemokines are a large subfamily of cytokines known for their ability to facilitate cell migration, most notably leukocytes, throughout the body. Chemokines are necessary for a functioning immune system in both health and disease and have received considerable attention for their roles in orchestrating temporal-spatial regulation of immune cell populations in cancer. Gliomas comprise a group of common central nervous system (CNS) primary tumors that are extremely challenging to treat. Immunotherapy approaches for highly malignant brain tumors offer an exciting new avenue for therapeutic intervention but so far, have seen limited successful clinical outcomes. Herein we focus on important chemokine/chemokine receptor systems in the regulation of pro- and anti-tumor mechanisms, highlighting potential therapeutic advantages of modulating these systems in malignant gliomas and other cancers.

The Stat3 inhibitor, S3I-201, downregulates lymphocyte activation markers, chemokine receptors, and inflammatory cytokines in the BTBR T+ Itpr3tf/J mouse model of autism.

Autism is a complex neurodevelopmental disorder with a high incidence rate. It is characterized by deficits in communication, a lack of social skills, cognitive inflexibility, and stereotypical behaviors. Autism has been gradually increasing in children over the past several years, without the existence of an effective treatment. BTBR T+ Itpr3tf/J (BTBR) mice serve as an accepted model to evaluate autistic-like behaviors as they display core behavioral symptoms displayed in autism. Previous findings showed that S3I-201, a selective Stat3 inhibitor, can be used to treat neuroinflammation disorders. Previously, we showed that S3I-201 treatment has therapeutic effects on autism-like behaviors, and Th1/Th17 and regulatory T cells in BTBR mice. The objective of the present study was to further explore the role of S3I-201 in BTBR mice, and this was performed by investigating the effects of S3I-201 treatment on lymphocyte activation markers (CD4+CD25+ and CD4+CD69+), chemokine receptors (CD4+CCR6+, CD4+CCR7+, CD4+CXCR4+, and CD4+CXCR5+), and proinflammatory cytokines (CD4+IL-6+ and CD4+TNF-α+) in the spleen cells of BTBR and C57BL/6 (C57) mice. The mRNA and protein expression levels of CD69, CCR6, CCR7, CXCR4, CXCR5, IL-1ß, IL-6, and TNF-α were examined in the brain tissues, and in BTBR mice, a significant decrease in CD25, CD69, CCR6, CCR7, CXCR4, CXCR5, IL-6, and TNF-α producing CD4+ T cells was observed. The present findings suggest that treatment with S3I-201 may be a therapeutic approach to improve immune abnormalities in a subgroup of autistic subjects.

The G-protein coupled receptor ChemR23 determines smooth muscle cell phenotypic switching to enhance high phosphate-induced vascular calcification.

AIMS: Vascular calcification, a marker of increased cardiovascular risk, is an active process orchestrated by smooth muscle cells. Observational studies indicate that omega-3 fatty acids protect against vascular calcification, but the mechanisms are unknown. The G-protein coupled receptor ChemR23 transduces the resolution of inflammation induced by the omega-3-derived lipid mediator resolvin E1. ChemR23 also contributes to osteoblastic differentiation of stem cells and bone formation, but its role in vascular calcification is unknown. The aim of this study was to establish the role of ChemR23 in smooth muscle cell fate and calcification. METHODS AND RESULTS: Gene expression analysis in epigastric arteries derived from patients with chronic kidney disease and vascular calcification revealed that ChemR23 mRNA levels predicted a synthetic smooth muscle cell phenotype. Genetic deletion of ChemR23 in mice prevented smooth muscle cell de-differentiation. ChemR23-deficient smooth muscle cells maintained a non-synthetic phenotype and exhibited resistance to phosphate-induced calcification. Moreover, ChemR23-deficient mice were protected against vitamin D3-induced vascular calcification. Resolvin E1 inhibited smooth muscle cell calcification through ChemR23. Introduction of the Caenorhabditis elegans Fat1 transgene, leading to an endogenous omega-3 fatty acid synthesis and hence increased substrate for resolvin E1 formation, significantly diminished the differences in phosphate-induced calcification between ChemR23+/+ and ChemR23-/- mice. CONCLUSION: This study identifies ChemR23 as a previously unrecognized determinant of synthetic and osteoblastic smooth muscle cell phenotype, favouring phosphate-induced vascular calcification. This effect may be of particular importance in the absence of ChemR23 ligands, such as resolvin E1, which acts as a calcification inhibitor under hyperphosphatic conditions.

Therapeutic Targeting of Chemokines and Chemokine Receptors in Multiple Sclerosis: Opportunities and Challenges.

BACKGROUND & OBJECTIVE: Multiple sclerosis is an autoimmune demyelinating disease of the human central nervous system with still unknown etiology. Infiltration, accumulation and activation of autoreactive T cells, macrophages and other inflammatory immune cells in the CNS are the crucial steps in MS neuropathogenesis and development. Chemokines and their receptors play the main role in the attraction of the pathogenic cells into the CNS in MS. Specific chemokines and chemokine receptors are up-regulated in the actively demyelinating lesions and cerebrospinal fluid of MS patients. Many medical studies investigated how changes in levels or activities of chemokines and their receptors are implicated in leukocyte migration into CNS and consequently causing MS. These chemokines and their receptors are under intense focus to introduce new therapeutic strategies for MS. CONCLUSION: The aim of this review is to summarize previous findings on the relationship between chemokines network and MS development. Furthermore, opportunities and challenges in the chemokine system intervention as a potential therapeutic target for the treatment of MS will be outlined.

Bisphenol A and its derivatives decrease expression of chemerin, which reverses its stimulatory action in ovarian cancer cells.

Chemerin is an adipocyte-secreted protein that associates with obesity, inflammation, metabolic dysfunction, and carcinogenesis. Previous studies have shown human granulosa cells to produce bioactive chemerin and its receptor CMKLR1. In the present study, we demonstrated that the mRNA level of chemerin receptor is higher in a granulosa cell tumor cell line than in epithelial cancer cells, whereas chemerin expression and secretion were lower. Various exogenous factors, such as bisphenol A and its halogenated derivatives tetrabromobisphenol A and tetrachlorobisphenol A, can affect adipokine expression. For this reason, we investigated the effects of bisphenol A and its derivatives on the expression of chemerin and its receptor. At low nanomolar concentrations, BPA, TBBPA, and TCBPA decreased chemerin expression and secretion only in granulosa cell tumor COV434 cells by both peroxisome proliferator-activated receptor γ and estrogen receptor signaling pathways. Chemerin treatment had no effect on proliferation of ovarian non-cancer and cancer cell lines. However, we also found evidence to support the inhibition of BPA- and TBBPA-induced cell proliferation by chemerin. Taken together, our results indicate for the first time that BPA and its derivatives down-regulate chemerin expression, which can suppress the ability of BPA to induce proliferation. Moreover, both PPARγ and ERs were involved in the BPA-induced decrease in chemerin expression, and its ratio was crucial to exert these effects.

Targeting the D Series Resolvin Receptor System for the Treatment of Osteoarthritis Pain.

OBJECTIVE: Pain is a major symptom of osteoarthritis (OA); currently available analgesics either do not provide adequate pain relief or are associated with serious side effects. The aim of this study was to investigate the therapeutic potential of targeting the resolvin receptor system to modify OA pain and pathology. METHODS: Gene expression of 2 resolvin receptors (ALX and ChemR23) was quantified in synovium and medial tibial plateau specimens obtained from patients with OA at the time of joint replacement surgery. Two models of OA joint pain were used for the mechanistic studies. Gene expression in the joint and central nervous system was quantified. The effects of exogenous administration of the D series resolvin precursor 17(R)-hydroxy-docosahexaenoic acid (17[R]-HDoHE) on pain behavior, joint pathology, spinal microglia, and astroglyosis were quantified. Plasma levels of relevant lipids, resolvin D2, 17(R)-HDoHE, and arachidonic acid, were determined in rats, using liquid chromatography tandem mass spectrometry. RESULTS: There was a positive correlation between resolvin receptor and interleukin-6 (IL-6) expression in human OA synovial and medial tibial plateau tissue. In rats, synovial expression of ALX was positively correlated with expression of IL-1ß, tumor necrosis factor, and cyclooxygenase 2. Treatment with 17(R)-HDoHE reversed established pain behavior (but not joint pathology) in 2 models of OA pain. This was associated with a significant elevation in the plasma levels of resolvin D2 and a significant reduction in astrogliosis in the spinal cord in the monosodium iodoacetate-induced OA rat model. CONCLUSION: Our preclinical data demonstrate the robust analgesic effects of activation of the D series resolvin pathways in 2 different animal models of OA. Our data support a predominant central mechanism of action in clinically relevant models of OA pain.

Novel Piperazino-Enaminones Suppress Pro-Inflammatory Cytokines and Inhibit Chemokine Receptor CCR2.

Pro-inflammatory mediators including TNF-alpha, IL-6, and nitric oxide are important for the regulation of the immune response when an infection is present, but when overproduced, it can be responsible for the development of tissue and organ injury seen in sepsis, as well as severe asthma, and autoimmune diseases such as Crohn's disease and rheumatoid arthritis. Data from our lab to characterize the novel compound enaminone E121 have suggested that macrophages stimulated with lipopolysaccharide (LPS) release significantly decreased levels of TNF-alpha and IL-6 as measured by enzyme-linked immunosorbent assay as compared to the DMSO control group. Additionally, functional experiments in a mouse model of asthma have shown that E121 is efficacious in decreasing airway hyperresponsiveness. A new set of compounds synthesized in our lab (JODI) have an N-aryl piperazino motif incorporated on the aromatic side of the enaminone pharmacophore. It was hypothesized that this would enhance their immunosuppressive activity as anti-inflammatory agents by also acting as a chemokine receptor antagonist. Our studies suggest that JODI appears to suppress TNF-alpha and IL-6 in a dose-dependent manner. The JODI compounds were also more effective in reducing TNF-alpha after LPS stimulation when compared to dexamethasone. Lastly, studies using MCP-1 suggest that the JODI compounds, and not E121, are able to block CCR2 signaling as evidenced by decreased total ERK1/2. These studies indicate that E121 and its corresponding piperazino analogs could act as strong anti-inflammatory agents in asthma or other autoimmunities where efficacious therapeutic options are needed.

A neuroendocrine role for chemerin in hypothalamic remodelling and photoperiodic control of energy balance.

Long-term and reversible changes in body weight are typical of seasonal animals. Thyroid hormone (TH) and retinoic acid (RA) within the tanycytes and ependymal cells of the hypothalamus have been implicated in the photoperiodic response. We investigated signalling downstream of RA and how this links to the control of body weight and food intake in photoperiodic F344 rats. Chemerin, an inflammatory chemokine, with a known role in energy metabolism, was identified as a target of RA. Gene expression of chemerin (Rarres2) and its receptors were localised within the tanycytes and ependymal cells, with higher expression under long (LD) versus short (SD) photoperiod, pointing to a physiological role. The SD to LD transition (increased food intake) was mimicked by 2 weeks of ICV infusion of chemerin into rats. Chemerin also increased expression of the cytoskeletal protein vimentin, implicating hypothalamic remodelling in this response. By contrast, acute ICV bolus injection of chemerin on a 12 h:12 h photoperiod inhibited food intake and decreased body weight with associated changes in hypothalamic neuropeptides involved in growth and feeding after 24 hr. We describe the hypothalamic ventricular zone as a key site of neuroendocrine regulation, where the inflammatory signal, chemerin, links TH and RA signaling to hypothalamic remodeling.

Common target genes of palatal and gingival fibroblasts for EMD: the microarray approach.

BACKGROUND AND OBJECTIVE: Connective tissue grafts are frequently applied, together with Emdogain(®) , for root coverage. However, it is unknown whether fibroblasts from the gingiva and from the palate respond similarly to Emdogain. The aim of this study was therefore to evaluate the effect of Emdogain(®) on fibroblasts from palatal and gingival connective tissue using a genome-wide microarray approach. MATERIAL AND METHODS: Human palatal and gingival fibroblasts were exposed to Emdogain(®) and RNA was subjected to microarray analysis followed by gene ontology screening with Database for Annotation, Visualization and Integrated Discovery functional annotation clustering, Kyoto Encyclopedia of Genes and Genomes pathway analysis and the Search Tool for the Retrieval of Interacting Genes/Proteins functional protein association network. Microarray results were confirmed by quantitative RT-PCR analysis. RESULTS: The transcription levels of 106 genes were up-/down-regulated by at least five-fold in both gingival and palatal fibroblasts upon exposure to Emdogain(®) . Gene ontology screening assigned the respective genes into 118 biological processes, six cellular components, eight molecular functions and five pathways. Among the striking patterns observed were the changing expression of ligands targeting the transforming growth factor-beta and gp130 receptor family as well as the transition of mesenchymal epithelial cells. Moreover, Emdogain(®) caused changes in expression of receptors for chemokines, lipids and hormones, and for transcription factors such as SMAD3, peroxisome proliferator-activated receptor gamma and those of the ETS family. CONCLUSION: The present data suggest that Emdogain(®) causes substantial alterations in gene expression, with similar patterns observed in palatal and gingival fibroblasts.

Chemokine receptors as therapeutic targets.

Chemokines are a group of structurally related secretory and transmembrane proteins whose major tasks are to coordinately recruit various leukocyte populations into target tissue sites via specific receptors. In humans, there are close to 50 chemokines, 18 signal transducing receptors and 5 decoy/scavenger receptors. Functionally, chemokines are grouped into two major categories. Inflammatory chemokines are those attracting and activating cells such as neutrophils, monocytes, and eosinophils, and thus play major roles in acute-type inflammatory conditions. They are characterized by high ligand redundancy and receptor promiscuity. This probably enables robust recruitment of inflammatory cells in acute conditions. On the other hand, immune chemokines are those mainly attracting lymphoid cells and dendritic cells, and are thus involved in immune responses and chronic inflammatory diseases. Furthermore, their ligand-receptor relationships are relatively monogamous. Chemokine receptors are all seven-transmembrane G protein-couple receptors, the class of receptors frequently targeted by many successful drugs. Thus, chemokine receptors are considered to be highly promising drug targets for inflammatory and immunological diseases, and for the last two decades, many pharmaceutical companies have been trying to develop drugs blocking specific chemokine receptors. However, there are only few instances that have reached the approval for clinical use. There are several possible reasons for the present stalemate. For example, the intrinsic functional redundancy in the chemokine system may have made blocking a single receptor useless. Furthermore, the unprecedented species differences even between humans and mice may have caused problems in determination of clinical application of each chemokine receptor blockade from animal studies and also in conducting preclinical studies of candidate drugs in animals. Thus, the potential of the chemokine system as drug targets may still remain underexplored. This review first overviews current potential clinical applications of individual chemokine receptors and then describes in detail the drugs now in clinical use : Maraviroc (CCR5 antagonist), Plerixafor (CXCR4 antagonist), and Mogamulizmab (anti-CCR4).

Emerging biomarkers and intervention targets for immune-modulation of atherosclerosis - a review of the experimental evidence.

The role of inflammation in atherosclerosis and plaque vulnerability is well recognized. However, it is only during recent years it has become evident that this inflammation is modulated by immune responses against plaque antigens such as oxidized LDL. Interestingly, both protective and pathogenic immune responses exist and experimental data from animal studies suggest that modulation of these immune responses represents a promising new target for treatment of cardiovascular disease. It has been proposed that during early stages of the disease, autoimmune responses against plaque antigens are controlled by regulatory T cells that inhibit the activity of auto-reactive Th1 effector T cells by release of anti-inflammatory cytokines such as IL-10 and TGF-ß. As the disease progresses this control is gradually lost and immune responses towards plaque antigens switch towards activation of Th1 effector T cells and release of pro-inflammatory cytokines such as interferon-γ, TNF-α and IL-1ß. Several novel immune-modulatory therapies that promote or mimic tolerogenic immune responses against plaque antigens have demonstrated athero-protective effects in experimental models and a first generation of such immune-modulatory therapies are now in early or about to enter into clinical testing. A challenge in the clinical development of these therapies is that our knowledge of the role of the immune system in atherosclerosis largely rests on data from animal models of the disease. It is therefore critical that more attention is given to the characterization and evaluation of immune biomarkers for cardiovascular risk.

Effect of S-aspirin, a novel hydrogen-sulfide-releasing aspirin (ACS14), on atherosclerosis in apoE-deficient mice.

Hydrogen sulfide (H(2)S) is a novel gaseous mediator that plays important roles in atherosclerosis. The present study investigated the effect of a novel H(2)S-releasing aspirin, ACS14 (2-acetyloxybenzoic acid 4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl ester), on atherosclerotic plaques in fat-fed apoE(-/-) mice and the underlying mechanism with respect to CX3C chemokine receptor 1 (CX3CR1) in macrophages. Mouse macrophage cell line RAW264.7 or mouse peritoneal macrophages were preincubated with aspirin (50, 100 or 200µM), ACS14 (50, 100 or 200µM) or vehicle for 6h, and then stimulated with interferon (IFN)-γ (500U/ml) or lipopolysaccharide (LPS; 10µg/ml) for 12h. ACS14, but not aspirin, dose-dependently inhibited IFN-γ or LPS-induced CX3CR1 expression and CX3CR1-mediated chemotaxis in macrophages. The inhibitory effect of ACS14 on CX3CR1 expression was abolished by pretreatment with GW9662, a selective peroxisome proliferator-activated receptor (PPAR)-γ antagonist, suggesting that suppression of macrophage CX3CR1 expression by ACS14 is PPAR-γ dependent. Eight-week-old male apoE(-/-) mice received intraperitoneal ACS14 (15 or 30µmol/kg/day) or aspirin (15 or 30µmol/kg/day) 4 weeks after fat feeding. Twelve weeks after ACS14 or aspirin treatment, mice were sacrificed to evaluate the extent of atherosclerosis and CX3CR1 expression in brachiocephalic artery (BCA). We found that ACS14, but not aspirin, significantly downregulated CX3CR1 expression in atherosclerotic plaques. ACS14 considerably impeded the formation and development of atherosclerosis as compared to a molar equivalent dose of aspirin. These data indicate that ACS14 may prevent the progression of atherosclerosis by downregulating macrophage CX3CR1 expression via a PPAR-γ-dependent mechanism.

RANTES-mediated control of excitatory amino acid release in mouse spinal cord.

The impact of Regulated upon Activation Normal T cells Expressed and Secreted (RANTES) on the release of pre-loaded [³H]D-aspartate ([³H]D-ASP) from mouse spinal cord synaptosomes was investigated. RANTES (0.01-1 nM) failed to affect the spontaneous release, but facilitated the 15 mM K⁺-evoked overflow of [³H]D-ASP. Incubation of synaptosomes with antibodies raised against the chemokine receptor (CCR)1 and CCR5 proteins prevented RANTES-induced facilitation of glutamate exocytosis, whereas anti-CCR3 antibody was inefficacious. Accordingly, BX513 and D-Ala-peptide T-amide (DAPTA) CCR1 and CCR5 antagonists, respectively, prevented RANTES-induced effect, whereas the CCR3 antagonist SB 328437 was inactive. To compare these findings to previous results, we quantified the effects of CCR antagonists on the RANTES-induced modifications of the spontaneous and the K⁺-evoked [³H]D-ASP release in the mouse cortex. Here, CCR1 and CCR5, but not CCR3, antagonists prevented the RANTES-mediated [³H]D-ASP release, whereas RANTES-induced inhibition of the 12 mM K⁺-evoked [³H]D-ASP exocytosis was also antagonized by SB 328437. Facilitation of glutamate exocytosis in spinal cord relied on PLC-dependent mobilization of Ca²âº from IP3-sensitive stores; adenylyl cyclase was not involved. CCR1, CCR3 and CCR5 receptor proteins were present in spinal cord synaptosomal and gliosomal lysates, although RANTES-induced changes to glutamate release could not be observed in gliosomes. Our results confirm the role of RANTES as modulator of glutamate transmission.

[The viral acceptor antagonist to entrapment ligand H9 in vitro activeness and the function to human acceptor CX3-CR1].

AIM: To clarify the activeness of H9 in vitro and internalization and modulation of the surface chemokine receptor CX3CR1 induced by H9, To discuss the influence of H9 on the chemokine receptor CX3CR1. METHODS: Inhibition by chemotactic peptide on the physiological detection of chemokine induced cell migration activity. Flowcytometry examined the effection of H9 on intracellular calcium. Laser scanning confocal microscopy and flow cytometry were used to determine the quality and quantity of CX3CR1 internalization. RESULTS: H9 was able to block the migration induced by chemokine receptor. In the chemoattraction test, H9 was unable to induce the chemotactic movement, and it does not affect the signal transduction and activeness of cells. It was found that H9 could induce internalization with a maximal rate of 70%, at the concentration of 200 ng/mL. The internalized CX3CR1 molecules could recycled to the cell surface. CONCLUSION: H9 makes human CX3CR1 internalize. After internalizing, the CX3CR1 receptor recirculates the cell surface. It does not affect CX3CR1 physiology function. H9 could be used as a specificity anti-virus peptide.

Areca nut extracts enhance the development of CD11b(+) Gr-1(+) cells with the characteristics of myeloid-derived suppressor cells in antigen-stimulated mice.

BACKGROUND: Areca quid chewing is an etiological factor contributing to the development of oral cancer and pre-cancers, whose pathophysiology has been linked to inflammation and immune deterioration. Myeloid-derived suppressor cells (MDSC) play a key role in the regulation of immunity under certain pathological conditions, such as inflammation and cancer. As areca nut extracts (ANE) have been reported to induce a proinflammatory effect in antigen-stimulated mice, we hypothesized that ANE might enhance the development of MDSC. METHODS: Ovalbumin (OVA)-sensitized BALB/c mice were daily administered with ANE (5-50 mg/kg), polyphenol-enriched ANE (PANE; 25 mg/kg) or arecoline (5 mg/kg) by intraperitoneal injection for 10 doses. The mouse footpads were then subcutaneously challenged with OVA to induce local inflammatory responses. RESULTS: ANE and PANE treatment significantly increased the spleen index and the population of CD11b(+) Gr-1(+) cells in the spleen and peripheral blood, whereas arecoline was inactive. In addition, ANE and PANE treatment enhanced the expression of cytokines and enzymes associated with the immunosuppressive function of MDSC, including IL-10, arginase-I and iNOS in splenic CD11b(+) cells. Concordantly, ANE and PANE treatment augmented the infiltration of Gr-1(+) IL-10(+) cells in the footpads challenged with OVA. CONCLUSIONS: Our results suggested that areca nut constituents, in particular, polyphenols enhanced the development of myeloid-derived suppressor cells in vivo, which may be a critical mechanism linking inflammation and the compromised immunity reported to be associated with the pathophysiology of areca-related oral diseases.

Chemerin/ChemR23 signaling axis is involved in the endothelial protection by K(ATP) channel opener iptakalim.

AIM: To elucidate the modulation of the chemerin/ChemR23 axis by iptakalim-induced opening of K(ATP) channels and to determine the role of the chemerin/ChemR23 axis in the iptakalim-mediated endothelial protection. METHODS: Cultured rat aortic endothelial cells (RAECs) were used. Chemerin secretion and ChemR23 protein expression were investigated using Western blot analysis. The gene expression level of ChemR23 was examined with RT-PCR. In addition, the release of nitric oxide (NO) was measured with a nitric oxide assay. RESULTS: Homocysteine, uric acid, high glucose, or oxidized low-density lipoprotein (ox-LDL) down-regulated the chemerin secretion and ChemR23 gene/protein expression in RAECs as a function of concentration and time, which was reversed by pretreatment with iptakalim (1-10 µmol/L). Moreover, these effects of iptakalim were abolished in the presence of the K(ATP) channel antagonist glibenclamide (1 µmol/L). Both iptakalim and recombinant chemerin restored the impaired NO production in RAECs induced by uric acid, and the effects were abolished by anti-ChemR23 antibodies. CONCLUSION: Iptakalim via opening K(ATP) channels enhanced the endothelial chemerin/ChemR23 axis and NO production, thus improving endothelial function.

[Curcumin down-regulates CX3CR1 expression in spinal cord dorsal horn and DRG in neuropathic pain rats].

OBJECTIVE: To investigate the effects of curcumin on the behavior of chronic constrictive injury (CCI) rats and the CX3CR1 expression in spinal cord dorsal horn and dorsal root ganglia (DRG). METHOD: Seventy-two male SD rats were randomly divided into 4 groups: 1) Sham operation group (Sham); 2) Chronic constrictive injury group (CCI); 3) Curcumin treated group (Cur), administrated with curcumin 100 mg x kg(-1) x d(-1) ip for 14 days after CCI; 4) Solvent contrast group (SC), administrated with an equal volume of solvent for 14 days after CCI. Paw thermal withdrawal (PTWL) and paw mechanical withdrawal threshold (PMWT) were measured on 2 pre-operative and 1, 3, 5, 7, 10, 14 post-operative days respectively. The lumbar segments L4-5 of the spinal cord and the L4, L5 DRG were removed at 3, 7, 14 days after surgery. The expression of CX3CR1 was determined by immunohistochemical staining. RESULT: Compared with Sham group, PTWL and PMWT in CCI group were significantly lower on each post-operative day (P<0.01), which reached a nadir on the 3rd day after CCI (PTWL was 6.5 +/- 1.1, PMWT was 22.6 +/- 5.1), and the expression of CX3CR1 were markedly increased in spinal cord dorsal horn and DRG. In Cur group, PTWL were higher than in CCI group on 7, 10, 14 post-operative day (P<0.05), and PMWT were higher than those in CCI group on 10 and 14 post-operative day (P<0.05). The administration of curcumin could significantly attenuate the activation of CX3CR1 induced by CCI. CONCLUSION: The study suggests that curcumin ameliorates the CCI-induced neuropathic pain, probably by attenuating the expression of CX3CR1 in spinal cord dorsal horn and dorsal root ganglia.

Emerging concepts and approaches for chemokine-receptor drug discovery.

IMPORTANCE OF THE FIELD: Chemokine receptors are most noted for their role in cell migration. However, inappropriate utilization or regulation of these receptors is implicated in many inflammatory diseases, cancer and HIV, making them important drug targets. AREAS COVERED IN THIS REVIEW: Allostery, oligomerization and ligand bias are presented as they pertain to chemokine receptors and their associated pathologies.Specific examples of each are described from the recent literature and their implications are discussed in terms of drug discovery efforts targeting chemokine receptors. WHAT THE READER WILL GAIN: Insight into the expanding view of the multitude of pharmacological variables that need to be considered or that may be exploited in chemokine receptor drug discovery. TAKE HOME MESSAGE: Since 2007, two drugs targeting chemokine receptors have been approved by the FDA, Maraviroc for preventing HIV infection and Mozobil™ for hematopoietic stem cell mobilization. While these successes permit optimism for chemokine receptors as drug targets, only recently has the complexity of this system begun to be appreciated. The concepts of allosteric inhibitors, biased ligands and functional selectivity raise the possibility that drugs with precisely-defined properties can be developed. Other complexities such as receptor oligomerization and tissue-specific functional states of receptors also offer opportunities for increased target and response specificity, although it will be more challenging to translate these ideas into approved therapeutics compared to traditional approaches.

Intrathecal infusion of pyrrolidine dithiocarbamate for the prevention and reversal of neuropathic pain in rats using a sciatic chronic constriction injury model.

BACKGROUND AND OBJECTIVES: Recent studies have suggested that nuclear factor κB (NF-κB) may play a role in mediating nerve injury-induced neuropathic pain. Here, we examined the effects of intrathecal pyrrolidine dithiocarbamate (PDTC), a NF-κB inhibitor, on the development of neuropathic pain, spinal microglial activation, and CX3CR1 expression induced by sciatic chronic constriction injury (CCI) model in rats. METHODS: Under chloral hydrate anesthesia, male Sprague-Dawley rats (300-350 g) fitted with intrathecal catheters underwent either sciatic CCI or sham surgery. Intrathecal saline or PDTC (100 or 1000 pmol/d) was infused 1 day before or 3 days after CCI (n = 8). The rat hind-paw withdrawal threshold to mechanical stimuli and withdrawal latency to radiant heat were determined before surgery and from days 1 to 7 after CCI. Spinal microglial activation was evaluated with OX-42 immunoreactivity, and spinal CX3CR1 expression was assessed by Western blotting. RESULTS: Chronic constriction injury induced mechanical allodynia and thermal hyperalgesia and microglial activation as demonstrated by OX-42 expression. Whereas it had no apparent effect on spinal cord histology, intrathecal administration of PDTC prevented the development of the mechanical and thermal hyperalgesia and inhibited nerve injury-induced microglial activation and spinal CX3CR1 expression. CONCLUSIONS: In this study, we have shown the protective effect of intrathecal PDTC on the development of nociceptive behaviors induced by CCI in rats. The activation of NF-κB pathway may contribute to spinal microglial activation and CX3CR1 up-regulation.