ChemR23 signaling ameliorates brain injury via inhibiting NLRP3 inflammasome-mediated neuronal pyroptosis in ischemic stroke.

BACKGROUND: Inflammatory response has been recognized as a pivotal pathophysiological process during cerebral ischemia. ChemR23 signaling is involved in the pathophysiology of various inflammatory diseases. Nevertheless, the role of ChemR23 signaling in ischemic stroke remains largely unknown. METHODS: Permanent ischemic stroke mouse model was accomplished by middle cerebral artery occlusion (MCAO). Resolvin E1 (RvE1) or chemerin-9 (C-9), the agonists of ChemR23, were administered by intracerebroventricular (i.c.v) injection before MCAO induction. Then, analysis of neurobehavioral deficits and brain sampling were done at Day 1 after MCAO. The brain samples were further analyzed by histological staining, immunofluorescence, RNA sequencing, ELISA, transmission electron microscope, and western blots. Furthermore, oxygen-glucose deprivation (OGD) was employed in SH-SY5Y to mimic MCAO in vitro, and ChemR23 signaling pathway was further studied by overexpression of ChemR23 or administration of related agonists or antagonists. Analysis of cell death and related pathway markers were performed. RESULTS: ChemR23 expression was upregulated following MCAO. Under in vitro and in vivo ischemic conditions, ChemR23 deficiency or inhibition contributed to excessive NLRP3-mediated maturation and release of IL-1ß and IL-18, as well as enhanced cleavage of GSDMD-N and neuronal pyroptosis. These influences ultimately aggravated brain injury and neuronal damage. On the other hand, ChemR23 activation by RvE1 or C-9 mitigated the above pathophysiological abnormalities in vivo and in vitro, and overexpression of ChemR23 in SH-SY5Y cells also rescued OGD-induced neuronal pyroptosis. Blockade of NLRP3 mimics the protective effects of ChemR23 activation in vitro. CONCLUSION: Our data indicated that ChemR23 modulates NLRP3 inflammasome-mediated neuronal pyroptosis in ischemic stroke. Activation of ChemR23 may serve as a promising potential target for neuroprotection in cerebral ischemia.

Effect of Regulation of Chemerin/Chemokine-like Receptor 1/Stimulator of Interferon Genes Pathway on Astrocyte Recruitment to Aß Plaques.

Recruitment and accumulation of reactive astrocytes around senile plaques are common pathological features of Alzheimer's disease (AD), with unclear mechanisms. Chemerin, an adipokine implicated in neuroinflammation, acts through its receptor, chemokine-like receptor 1 (CMKLR1), which also functions as a receptor for amyloid ß (Aß). The impact of the chemerin/CMKLR1 axis on astrocyte migration towards Aß plaques is unknown. Here we investigated the effect of CMKLR1 on astrocyte migration around Aß deposition in APP/PS1 mice with Cmklr1 knockout (APP/PS1-Cmklr1-/-). CMKLR1-expressed astrocytes were upregulated in the cortices and hippocampi of 9-month-old APP/PS1 mice. Chemerin mainly co-localized with neurons, and its expression was reduced in the brains of APP/PS1 mice, compared to WT mice. CMKLR1 deficiency decreased astrocyte colocalization with Aß plaques in APP/PS1-Cmklr1-/- mice, compared to APP/PS1 mice. Activation of the chemerin/CMKLR1 axis promoted the migration of primary cultured astrocytes and U251 cells, and reduced astrocyte clustering induced by Aß42. Mechanistic studies revealed that chemerin/CMKLR1 activation induced STING phosphorylation. Deletion of STING attenuated the promotion of the chemerin/CMKLR1 axis relative to astrocyte migration and abolished the inhibitory effect of chemerin on Aß42-induced astrocyte clustering. These findings suggest the involvement of the chemerin/CMKLR1/STING pathway in the regulation of astrocyte migration and recruitment to Aß plaques/Aß42.

Association of Single Nucleotide Polymorphisms (SNPs) of Chemoattractant Receptor23 (ChemR23) Gene with Susceptibility to Allergic Rhinitis.

The chemoattractant Receptor23 (ChemR23) plays an essential role in triggering and resolving acute inflammation. This study aimed to evaluate the association between four potentially functional SNPs of the chemR23 gene (rs4373981 G > C, rs73201532 C > T, rs35121177 G > A, and rs4964676 G > A) with susceptibility to Allergic rhinitis (AR). 130 patients with allergic rhinitis and 130 healthy individuals were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Our findings showed that genotypes and alleles frequencies were not significantly different between patient and control groups (p > 0.05). Furthermore, haplotype analysis (rs4373981, rs73201532, and rs4964676, respectively) revealed a protective effect of CTG, GTA, and GTG haplotypes against AR (p = 0.009, p = 0.0001, p = 0.001, respectively), and CCG, GCA, and GCG haplotypes of ChemR23 polymorphisms were associated with increased risk of AR (p = 0.03, p = 0.02, p = 0.0002, respectively). These findings suggested a possible role for ChemR23 in the pathogenesis of AR.

Chemerin regulates normal angiogenesis and hypoxia-driven neovascularization.

Chemerin is a multifunctional protein initially characterized in our laboratory as a chemoattractant factor for leukocyte populations. Its main functional receptor is CMKLR1. We identified previously chemerin as an anti-tumoral factor inhibiting the vascularization of tumor grafts. We show here that overexpression of bioactive chemerin in mice results in a reduction of the density of the retinal vascular network during its development and in adults. Chemerin did not affect vascular sprouting during the post-natal development of the network, but rather promoted endothelial cell apoptosis and vessel pruning. This phenotype was reversed to normal in CMKLR1-deficient mice, demonstrating the role of this receptor. Chemerin inhibited also neoangiogenesis in a model of pathological proliferative retinopathy, and in response to hind-limb ischemia. Mechanistically, PTEN and FOXO1 antagonists could almost completely restore the density of the retinal vasculature, suggesting the involvement of the PI3-kinase/AKT pathway in the chemerin-induced vessel regression process.

Role of Chemerin/ChemR23 axis as an emerging therapeutic perspective on obesity-related vascular dysfunction.

Sufficient epidemiological investigations demonstrate that there is a close correlation between obesity and vascular dysfunction. Nevertheless, specific mechanisms underlying this link remain currently unclear. Given the crucial and decisive role of vascular dysfunction in multitudinous diseases, various hypotheses had been proposed and numerous experiments were being carried out. One recognized view is that increased adipokine secretion following the expanded mass of white adipose tissue due to obesity contributes to the regulation of vascular function. Chemerin, as a neo-adipokine, whose systemic level is elevated in obesity, is believed as a regulator of adipogenesis, inflammation, and vascular dysfunction via binding its cell surface receptor, chemR23. Hence, this review aims to focus on the up-to-date proof on chemerin/chemR23 axis-relevant signaling pathways, emphasize the multifarious impacts of chemerin/chemR23 axis on vascular function regulation, raise certain unsettled questions to inspire further investigations, and explore the therapeutic possibilities targeting chemerin/chemR23.

Discovery and mechanistic study of thiazole-4-acylsulfonamide derivatives as potent and orally active ChemR23 inhibitors with a long-acting effect in cynomolgus monkeys.

Plasmacytoid dendritic cells (pDCs) are a subset of dendritic cells that can secrete large amounts of type I interferon. ChemR23, a G protein-coupled receptor (GPCR) expressed on the surface of pDCs, contributes to the recruitment of pDCs to inflamed tissues through chemotaxis signaling, and is therefore considered an attractive target for the treatment of autoimmune diseases. We previously reported benzoxazole-based compounds that can inhibit ChemR23 signaling through receptor internalization. Although these compounds showed ChemR23 internalization on pDCs in cynomolgus monkeys after oral administration, further improvement of the pharmacokinetics profile was needed for a clinical candidate and we therefore attempted scaffold-hopping from the benzoxazole core structure leading to novel thiazole derivatives. In this report, the design, synthesis, and biological evaluation of new thiazole-based ChemR23 inhibitors were described. Through sequential structure-activity relationship studies regarding (i) the side chain of the N-acylsulfonamide moiety, (ii) the 5-position of the thiazole ring, and (iii) the 1,2,4-oxadiazol-5-one moiety, we have succeeded in finding a potent thiazole-based ChemR23 inhibitor, 14f (IC80 = 12 nM). In addition, the oral administration of 14f at 30 mg/kg to cynomolgus monkeys demonstrated a sustained pharmacological effect of ChemR23 internalization on pDCs until 8 h after dosing, which was considered a longer effect in comparison to previously reported 2-aminobenzoxazole-based ChemR23 inhibitors. This report also shows the synthesis and evaluation of fluorescein-labeled compound 45c for a mechanistic study, and we could confirm the direct binding of our thiazole derivative to ChemR23. We believe that our research on small molecule ChemR23 inhibitors and chemical probe will contribute to the elucidation and analysis of the functions of ChemR23 as well as identifying novel therapeutics for autoimmune diseases.

Photoluminescent carbon dots (PCDs) from sour apple: a biocompatible nanomaterial for preventing UHMWPE wear-particle induced osteolysis via modulating Chemerin/ChemR23 and SIRT1 signaling pathway and its bioimaging application.

Photoluminescent nanomaterials have been widely employed in several biological applications both in vitro and in vivo. For the first time, we report a novel application of sour apple-derived photoluminescent carbon dots (PCDs) for reducing ultra-high molecular weight polyethylene (UHMWPE) wear particle-induced osteolysis using mouse calvarial model. Generally, aseptic prosthetic loosening seems to be a significant postoperative problem for artificial joints replacement, which is mainly contributed by UHMWPE-induced osteolysis. Hence, inhibiting osteoclastic bone-resorption could minimize UHMWPE-induced osteolysis for implant loosening. Prior to osteolysis studies, the prepared sour apple-derived PCDs were employed for bioimaging application. As expected, the prepared PCDs effectively inhibited the UHMWPE particle-induced osteoclastogenesis in vitro. The PCDs treatment effectively inhibited the UHMWPE-induced osteoclast differentiation, F-actin ring pattern, and bone resorption in vitro. Also, the PCDs reduced the UHMWPE-induced ROS stress as well as the expression level of pro-inflammatory cytokines, including TNF-α, IL-1, IL-6, and IL-8. Further, the qPCR and western blot results hypothesized that PCDs inhibited the UHMWPE wear particle-induced osteolysis through suppressing chemerin/ChemR23 signaling and NFATc1 pathway, along with upregulation of SIRT1 expression. Overall, these findings suggest that the synthesized PCDs could be a potential therapeutic material for minimizing UHMWPE particle-induced periprosthetic osteolysis to avoid postoperative complications.

The Chemerin/CMKLR1 Axis Is Involved in the Recruitment of Microglia to Aß Deposition through p38 MAPK Pathway.

The accumulation of microglia around senile plaques is one of the pathological features of Alzheimer's disease (AD). Chemerin is an adipokine with immune-modulating properties. Our previous study showed that chemokine-like receptor 1 (CMKLR1), the receptor for chemerin, is also a functional receptor of Aß. However, it remains unclear whether and how the chemerin/CMKLR1 axis affects the migration of microglia. The impact of CMKLR1 on microglial activation and recruitment toward Aß deposits was examined in APP/PS1 mice mated with CMKLR1 knockout (CMKLR1-/-) mice. CMKLR1 deficiency reduced the number of microglia around Aß deposits in aged APP/PS1-CMKLR1-/- mice compared with APP/PS1 mice. Chemerin expression was significantly decreased in the hippocampus and cortex of aged APP/PS1 mice compared with WT mice. In vitro assays demonstrated that activation of the chemerin/CMKLR1 axis promoted the migration of primary cultures of microglia and murine microglial N9 cells. Mechanistic studies found that chemerin/CMKLR1 induced polarization and protrusion formation of microglia by promoting the remodeling of actin filaments and microtubules, and Golgi apparatus reorientation. The inhibition of p38 MAPK attenuated the promotion of the chemerin/CMKLR1 axis on microglial migration and polarization. In addition, chemerin inhibited Aß-induced microglial clustering. The inhibition of p38 MAPK alleviated the suppressive effect of chemerin on Aß-induced microglial aggregation. Our data indicate that the chemerin/CMKLR1 axis is involved in the migration and recruitment of microglia to senile plaques via the p38 MAPK pathway. Modulation of the chemerin/CMKLR1 axis is a potential new strategy for AD therapy.

The design, synthesis and evaluation of 2-aminobenzoxazole analogues as potent and orally efficacious ChemR23 inhibitors.

We previously reported 2-aminobenzoxazole analogue 1 as a potent ChemR23 inhibitor. The compound showed inhibitory activity against chemerin-induced calcium signaling through ChemR23 internalization in CAL-1 cells, which are cell lines of plasmacytoid dendric cells (pDCs). Furthermore, compound 2 inhibited chemotaxis of CAL-1 triggered by chemerin in vitro. However, we noted a difference in the ChemR23 response to our inhibitor between rodents and non-rodents in a previous study. To address this issue, we performed optimization of ChemR23 inhibitors using CAL-1 cells endogenously expressing human ChemR23 and conducted a pharmacokinetics study in cynomolgus monkeys. Various substituents at the 4-position of the benzoxazole ring exhibited potent in vitro bioactivity, while those at the 6-position were not tolerated. Among substituents, a carboxyl group was identified as key for improving the oral bioavailability in cynomolgus monkeys. Compound 38a with the acidic part changed from a tetrazole group to a 1,2,4-oxadiazol-5-one group to improve bioactivity and pharmacokinetic parameters exhibited inhibitory activity against chemerin-induced chemotaxis in vitro. In addition, we confirmed the ChemR23 internalization of pDCs by compound 38a orally administered to cynomolgus monkeys. These 2-aminobenzoxazole-based ChemR23 inhibitors may be useful as novel immunotherapeutic agents capable of suppressing the migration of pDCs, which are known to be major producers of type I interferons in the lesion area of certain autoimmune diseases, such as systemic lupus erythematosus and psoriasis.

Chemerin/ChemR23 axis promotes inflammation of glomerular endothelial cells in diabetic nephropathy.

Diabetic nephropathy (DN) is characterized by inflammation of renal tissue. Glomerular endothelial cells (GEnCs) play an important role in inflammation and protein leakage in urine in DN patients. Chemerin and its receptor ChemR23 are inducers of inflammation. The aim of this study was to investigate the function of chemerin/ChemR23 in GEnCs of DN patients. Immunohistochemical staining and qRT-PCR were used to measure the expression of chemerin, ChemR23 and inflammatory factors in renal tissues of DN patients. Db/db mice were used as animal model. ChemR23 of DN mice was knocked down by injecting LV3-shRNA into tail vein. Inflammation, physiological and pathological changes in each group was measured. GEnCs were cultured as an in vitro model to study potential signalling pathways. Results showed that expression of chemerin, ChemR23 and inflammatory factors increased in DN patients and mice. LV3-shRNA alleviated renal damage and inflammation in DN mice. GEnCs stimulated by glucose showed increased chemerin, ChemR23 and inflammatory factors and decreased endothelial marker CD31. Both LV3-shRNA and SB203580 (p38 MAPK inhibitor) attenuated chemerin-induced inflammation and injury in GEnCs. Taken together, chemerin/ChemR23 axis played an important role in endothelial injury and inflammation in DN via the p38 MAPK signalling pathway. Suppression of ChemR23 alleviated DN damage.

Chemerin-induced macrophages pyroptosis in fetal brain tissue leads to cognitive disorder in offspring of diabetic dams.

BACKGROUND: Chemerin is highly expressed in the serum, placenta tissue, and umbilical cord blood of diabetic mother; however, the impact of chemerin on cognitive disorders of offspring from mothers with diabetes in pregnancy remains unclear. METHODS: A diabetic phenotype in pregnant mice dams was induced by streptozocin (STZ) injection or intraperitoneal injection of chemerin. Behavioral changes in offspring of diabetic dams and nondiabetic controls were assessed, and changes in chemerin, two receptors of chemerin [chemerin receptor 23 (ChemR23) and chemokine (C-C motif) receptor-like 2 (CCRL2)], macrophages, and neurons in the brain tissue were studied to reveal the underlying mechanism of the behavioral changes. RESULTS: Chemerin treatment mimicked the STZ-induced symptom of maternal diabetes in mice along with the altered behavior of offspring in the open field test (OFT) assay. In the exploring process for potential mechanism, the brain tissues of offspring from chemerin-treated dams were observed with an increase level of macrophage infiltration and a decrease number of neuron cells. Moreover, an increased level of NOD-like receptor family pyrin domain containing 3 (NLRP3) and apoptosis-associated speck-like (Asc) protein as well as pyroptosis [characterized by increased active caspase-1 content and secretion of cytokines such as interleukin (IL) 1 beta (IL-1ß) and IL-18] more activated in macrophages is also observed in the brain of these diabetic dam's offspring, in the presence of ChemR23. In vitro, it was found that pyroptosis activation was increased in macrophages separated from the abdominal cavity of normal mice, after chemerin treatment. However, depletion of CCRL2 decreased the level of chemerin in the brain tissues of diabetic dams' offspring; depletion of ChemR23 decreased macrophage pyroptosis, and depletion of either receptor reversed chemerin-mediated neurodevelopmental deficits and cognitive impairment of offspring of diabetic pregnant dams. CONCLUSIONS: Chemerin induced diabetic pregnant disease and CCRL2 were required to enrich chemerin in the brain of offspring. Aggregation of chemerin could lead to macrophage recruitment, activation of pyroptosis, the release of inflammatory cytokines, a decrease in the number of neurons, and cognitive impairment in offspring in a ChemR23-dependent manner. Targeting CCRL2 and/or ChemR23 could be useful for treating neuropsychological deficits in offspring of dams with diabetes in pregnancy.

The discovery and optimization of a series of 2-aminobenzoxazole derivatives as ChemR23 inhibitors.

A structural class of 2-aminobenzoxazole derivatives possessing biphenyltetrazole was discovered to be potent human ChemR23 inhibitors. We initially tried to improve the potency of compound 1, which was found through in-house screening using the human plasmacytoid dendritic cell (pDC)-like cell line CAL-1. The introduction of a chiral methyl moiety at a benzylic position in a center of compound 1 showed a large impact on the inhibitory activity against calcium signaling of ChemR23 induced by the natural ligand chemerin. As a result of further investigations at the benzylic position, (R)-isomer 6b was found to show a 30-fold increased potency over desmethyl compound 1. In addition, an extensive structure-activity relationship study on the benzoxazole moiety successfully led to a further increase in the potency. The antagonistic effect of the compounds was based on the induction of ChemR23 internalization. In addition, we observed that compound 31, which contained an amide moiety on benzoxazole, inhibited chemotaxis of CAL-1 cells induced by chemerin in vitro. These results suggest that our ChemR23 inhibitors are attractive compounds for the treatment of pDC-related autoimmune diseases, such as systemic lupus erythematosus and psoriasis.

Evaluation of chemerin and its receptors, ChemR23 and CCRL2, in gingival tissues with healthy and periodontitis.

Chemerin is a chemoattractant protein that directs inflammatory cells that express its receptor chemokine receptor-like 1 (ChemR23) towards sites of inflammation. C-C chemokine receptor-like 2 (CCRL2), is the other receptor of chemerin, improves the interaction between chemerin and ChemR23. The aim of this study was to evaluate the expression of chemerin and its receptors in gingival tissues with healthy and periodontitis. Tissue biopsy samples were obtained from 20 patients with chronic periodontitis and from the gingiva of 20 healthy individuals undergoing a crown lengthening process. Quantitative real-time PCR (qPCR) was used to examine the mRNA expression of chemerin, ChemR23 and CCRL2. Additionally, protein expression was measured by immunohistochemistry. Both qPCR and immunohistochemistry results revealed that the expression of chemerin and ChemR23 was significantly higher in tissues with periodontitis than in healthy tissues (P = 0.001 and, P = 0.015, respectively). There were no significant differences between healthy tissues and those with periodontitis in terms of mRNA expression of CCRL2, whereas a more intense staining was observed in tissues with periodontitis. The mRNA expression levels of chemerin showed a positive correlation with plaque index, gingival index, probing pocket depth and clinical attachment level (r = 0.448, r = 0.460, r = 0.439 and, r = 0.459, respectively, P < 0.01). To the best of our knowledge, this study is the first to examine the expression of chemerin, ChemR23 and CCRL2 in gingival tissues. Our study suggests that chemerin may play a role in the pathogenesis of periodontitis by causing chemoattraction of immune cells that direct ChemR23 receptors to the site of inflammation.

Upregulation of Nrf2 and Decreased Redox Signaling Contribute to Renoprotective Effects of Chemerin Receptor Blockade in Diabetic Mice.

Chemerin, acting through its receptor ChemR23, is an adipokine associated with inflammatory response, glucose and lipid metabolism and vascular function. Although this adipokine has been associated with the development and progression of kidney disease, it is not clear whether the chemerin/ChemR23 system plays a role in renal function in the context of diabetes. Therefore, we sought to determine whether ChemR23 receptor blockade prevents the development and/or progression of diabetic nephropathy and questioned the role of oxidative stress and Nrf2 in this process. Renal redox state and function were assessed in non-diabetic lean db/m and diabetic obese db/db mice treated with vehicle or CCX832 (ChemR23 antagonist). Renal reactive oxygen species (ROS) production, which was increased in diabetic mice, was attenuated by CCX832. This was associated with an increase in Nox 4 expression. Augmented protein oxidation in db/db mice was not observed when mice were treated with CCX832. CCX832 also abrogated impaired Nrf2 nuclear activity and associated downregulation in antioxidants expression in kidneys from db/db mice. Our in vivo findings highlight the role of the redox signaling and Nrf2 system as renoprotective players during chemerin receptor blockade in diabetic mice. The chemerin/ChemR23 system may be an important target to limit renal dysfunction associated with obesity-related diabetes.

Mesenchymal stromal cell-secreted chemerin is a novel immunomodulatory molecule driving the migration of ChemR23-expressing cells.

BACKGROUND: Mesenchymal stromal cells (MSCs) are multipotent cells characterized by broad immunomodulatory properties exploited for the treatment of inflammatory disorders. However, the efficacy of MSC-based therapy is highly variable and tightly linked to MSC culture conditions and treatment schedule. Thus, the identification of novel key molecules regulating MSC immunomodulatory activities in vivo might constitute a crucial step toward the optimization of currently available clinical protocols. In this regard, herein, we sought to determine whether the newly identified chemotactic protein, chemerin, plays a role in MSC-mediated regulation of inflammation. METHODS: Chemerin production by human MSCs was investigated under different culture conditions using enzyme-linked immunosorbent assay (ELISA). After purification, MSC-secreted chemerin was identified using mass spectrometry analysis and the biological activity of secreted isoforms was evaluated using migration assay. RESULTS: Bone marrow-derived MSCs secrete chemerin and express its receptors ChemR23 and CCRL2. Chemerin production is dependent on culture conditions and increases upon stimulation with inflammatory cytokines. In particular, platelet lysate (PL)-MSCs produce higher levels of chemerin compared with fetal bovine serum (FBS)-MSCs. Furthermore, chemerin is secreted by MSCs as an inactive precursor, which can be converted into its active form by exogenous chemerin-activating serine and cysteine proteases. DISCUSSION: Our data indicate that, in response to various inflammatory stimuli, MSCs secrete high amounts of inactive chemerin, which can then be activated by inflammation-induced tissue proteases. In light of these initial findings, we propose that further analysis of chemerin functions in vivo might constitute a crucial step toward optimizing MSC-based therapy for inflammatory diseases.

The adipokine chemerin amplifies electrical field-stimulated contraction in the isolated rat superior mesenteric artery.

The adipokine chemerin causes arterial contraction and is implicated in blood pressure regulation, especially in obese subjects with elevated levels of circulating chemerin. Because chemerin is expressed in the perivascular adipose tissue (PVAT) that surrounds the sympathetic innervation of the blood vessel, we tested the hypothesis that chemerin (endogenous and exogenous) amplifies the sympathetic nervous system in mediating electrical field-stimulated (EFS) contraction. The superior mesenteric artery, with or without PVAT and with endothelium and sympathetic nerve intact, was mounted into isolated tissue baths and used for isometric contraction and stimulation. Immunohistochemistry validated a robust expression of chemerin in the PVAT surrounding the superior mesenteric artery. EFS (0.3-20 Hz) caused a frequency-dependent contraction in isolated arteries that was reduced by the chemerin receptor ChemR23 antagonist CCX832 alone (100 nM; with, but not without, PVAT), but not by the inactive congener CCX826 (100 nM). Exogenous chemerin-9 (1 µM)-amplified EFS-induced contraction in arteries (with and without PVAT) was blocked by CCX832 and the α-adrenergic receptor antagonist prazosin. CCX832 did not directly inhibit, nor did chemerin directly amplify, norepinephrine-induced contraction. Whole mount immunohistochemical experiments support colocalization of ChemR23 with the sympathetic nerve marker tyrosine hydroxylase in superior mesenteric PVAT and, to a lesser extent, in arteries and veins. These studies support the idea that exogenous chemerin modifies sympathetic nerve-mediated contraction through ChemR23 and that ChemR23 may be endogenously activated. This is significant because of the well-appreciated role of the sympathetic nervous system in blood pressure control.

Development of a membrane-anchored chemerin receptor agonist as a novel modulator of allergic airway inflammation and neuropathic pain.

The chemerin receptor (CMKLR1) is a G protein-coupled receptor found on select immune, epithelial, and dorsal root ganglion/spinal cord neuronal cells. CMKLR1 is primarily coupled to the inhibitory G protein, Gαi, and has been shown to modulate the resolution of inflammation and neuropathic pain. CMKLR1 is activated by both lipid and peptide agonists, resolvin E1 and chemerin, respectively. Notably, these ligands have short half-lives. To expedite the development of long acting, stable chemerin analogs as candidate therapeutics, we used membrane-tethered ligand technology. Membrane-tethered ligands are recombinant proteins comprised of an extracellular peptide ligand, a linker sequence, and an anchoring transmembrane domain. Using this technology, we established that a 9-amino acid-tethered chemerin fragment (amino acids 149-157) activates both mouse and human CMKLR1 with efficacy exceeding that of the full-length peptide (amino acids 21-157). To enable in vivo delivery of a corresponding soluble membrane anchored ligand, we generated lipidated analogs of the 9-amino acid fragment. Pharmacological assessment revealed high potency and wash resistance (an index of membrane anchoring). When tested in vivo, a chemerin SMAL decreased allergic airway inflammation and attenuated neuropathic pain in mice. This compound provides a prototype membrane-anchored peptide for the treatment of inflammatory disease. A parallel approach may be applied to developing therapeutics targeting other peptide hormone G protein-coupled receptors.

Resolution of inflammation is altered in Alzheimer's disease.

BACKGROUND: Resolution is the final stage of the inflammatory response, when restoration of tissue occurs. Failure may lead to chronic inflammation, which is known as part of the pathology in the brain of individuals with Alzheimer's disease (AD). METHODS: Specialized pro-resolving mediators (SPMs), receptors, biosynthetic enzyme, and downstream effectors involved in resolution were analyzed in postmortem hippocampal tissue from AD patients and non-AD subjects. SPMs were analyzed in cerebrospinal fluid (CSF). RESULTS: SPMs and SPM receptors were detected in the human brain. Levels of the SPM lipoxin A4 (LXA4) were reduced in AD, both in the CSF and hippocampus. An enzyme involved in LXA4 synthesis and two SPM receptors were elevated in AD brains. LXA4 and RvD1 levels in CSF correlated with Mini-Mental State Examination (MMSE) scores. CONCLUSIONS: A resolution pathway exists in the brain and the alterations described herein strongly suggest a dysfunction of this pathway in AD. MMSE correlations suggest a connection with cognitive function in AD.

Chemerin connects fat to arterial contraction.

OBJECTIVE: Obesity and hypertension are comorbid in epidemic proportion, yet their biological connection is largely a mystery. The peptide chemerin is a candidate for connecting fat deposits around the blood vessel (perivascular adipose tissue) to arterial contraction. We presently tested the hypothesis that chemerin is expressed in perivascular adipose tissue and is vasoactive, supporting the existence of a chemerin axis in the vasculature. APPROACH AND RESULTS: Real-time polymerase chain reaction, immunohistochemistry, and Western analyses supported the synthesis and expression of chemerin in perivascular adipose tissue, whereas the primary chemerin receptor ChemR23 was expressed both in the tunica media and endothelial layer. The ChemR23 agonist chemerin-9 caused receptor, concentration-dependent contraction in the isolated rat thoracic aorta, superior mesenteric artery, and mesenteric resistance artery, and contraction was significantly amplified (more than 100%) when nitric oxide synthase was inhibited and the endothelial cell mechanically removed or tone was placed on the arteries. The novel ChemR23 antagonist CCX832 inhibited phenylephrine-induced and prostaglandin F2α-induced contraction (+perivascular adipose tissue), suggesting that endogenous chemerin contributes to contraction. Arteries from animals with dysfunctional endothelium (obese or hypertensive) demonstrated a pronounced contraction to chemerin-9. Finally, mesenteric arteries from obese humans demonstrate amplified contraction to chemerin-9. CONCLUSIONS: These data support a new role for chemerin as an endogenous vasoconstrictor that operates through a receptor typically attributed to function only in immune cells.

Resolvins E1 and D1 inhibit interstitial fibrosis in the obstructed kidney via inhibition of local fibroblast proliferation.

Resolvin E1 (RvE1) is a naturally occurring lipid-derived mediator generated during the resolution of inflammation. The anti-inflammatory effects of RvE1 have been demonstrated in a variety of disease settings; however, it is not known whether RvE1 may also exert direct anti-fibrotic effects. We examined the potential anti-fibrotic actions of RvE1 in the mouse obstructed kidney-a model in which tissue fibrosis is driven by unilateral ureteric obstruction (UUO), an irreversible, non-immune insult. Administration of RvE1 (300 ng/day) to mice significantly reduced accumulation of α-smooth muscle actin (SMA)(+) myofibroblasts and the deposition of collagen IV on day 6 after UUO. This protective effect was associated with a marked reduction of myofibroblast proliferation on days 2, 4 and 6 after UUO. RvE1 treatment also inhibited production of the major fibroblast mitogen, platelet-derived growth factor-BB (PDGF-BB), in the obstructed kidney. Acute resolvin treatment over days 2-4 after UUO also had a profound inhibitory effect upon myofibroblast proliferation without affecting the PDGF expression, suggesting a direct effect upon fibroblast proliferation. In vitro studies established that RvE1 can directly inhibit PDGF-BB-induced proliferation in primary mouse fibroblasts. RvE1 induced transient, but not sustained, activation of the pro-proliferative ERK and AKT signalling pathways. Of note, RvE1 inhibited the sustained activation of ERK and AKT pathways seen in response to PDGF stimulation, thereby preventing up-regulation of molecules required for progression through the cell cycle (c-Myc, cyclin D) and down-regulation of inhibitors of cell cycle progression (p21, cip1). Finally, siRNA-based knock-down studies showed that the RvE1 receptor, ChemR23, is required for the anti-proliferative actions of RvE1 in cultured fibroblasts. In conclusion, this study demonstrates that RvE1 can inhibit fibroblast proliferation in vivo and in vitro, identifying RvE1 as a novel anti-fibrotic therapy.