Targeting Interleukin-1ß Protects from Aortic Aneurysms Induced by Disrupted Transforming Growth Factor ß Signaling.

Aortic aneurysms are life-threatening conditions with effective treatments mainly limited to emergency surgery or trans-arterial endovascular stent grafts, thus calling for the identification of specific molecular targets. Genetic studies have highlighted controversial roles of transforming growth factor ß (TGF-ß) signaling in aneurysm development. Here, we report on aneurysms developing in adult mice after smooth muscle cell (SMC)-specific inactivation of Smad4, an intracellular transducer of TGF-ß. The results revealed that Smad4 inhibition activated interleukin-1ß (IL-1ß) in SMCs. This danger signal later recruited innate immunity in the adventitia through chemokine (C-C motif) ligand 2 (CCL2) and modified the mechanical properties of the aortic wall, thus favoring vessel dilation. SMC-specific Smad4 deletion in Il1r1- or Ccr2-null mice resulted in milder aortic pathology. A chronic treatment with anti-IL-1ß antibody effectively hampered aneurysm development. These findings identify a mechanistic target for controlling the progression of aneurysms with compromised TGF-ß signaling, such as those driven by SMAD4 mutations.

A CCL2/MCP-1 antagonist attenuates fibrosis of the infrapatellar fat pad in a rat model of arthritis.

BACKGROUND: Fibrosis of the infrapatellar fat pad (IFP) is a feature of osteoarthritis and contributes substantially to the pain and dysfunction in patients' joints. However, the underlying mechanisms remain unclear. C-C motif chemokine ligand-2 (CCL2) plays a central role in tissue fibrosis. Thus, we aimed to investigate the role of CCL2 in the development of IFP fibrosis in a rat model of arthritis, hypothesizing that a CCL2 antagonist could mitigate fibrotic progression. METHODS: We induced arthritis in male Wistar rats using intra-articular injections of carrageenan. Furthermore, to evaluate the effects of a CCL2 antagonist on protein expression and collagen deposition in the IFP of the rats, we transferred an N-terminal-truncated CCL2 gene into a rat model via electroporation-mediated intramuscular injection. Macrophage infiltration and collagen deposition in the IFP were analyzed in vivo. Groups were compared using the Mann-Whitney U test and Student's t-test. RESULTS: We identified infiltrating macrophages as well as increases in CCL2 and TGF-ß levels as collagen deposition progressed. Gene transfer of the CCL2-antagonist before arthritis induction attenuated collagen deposition remarkably. CONCLUSIONS: We provide initial evidence that anti-CCL2 gene therapy can effectively suppress the development of IFP fibrosis in a rat model. Thus, targeting CCL2 holds promise as a therapeutic strategy for managing tissue fibrosis in osteoarthritis patients.

ROS-induced PADI2 downregulation accelerates cellular senescence via the stimulation of SASP production and NFκB activation.

Cellular senescence is closely related to tissue aging including bone. Bone homeostasis is maintained by the tight balance between bone-forming osteoblasts and bone-resorbing osteoclasts, but it undergoes deregulation with age, causing age-associated osteoporosis, a main cause of which is osteoblast dysfunction. Oxidative stress caused by the accumulation of reactive oxygen species (ROS) in bone tissues with aging can accelerate osteoblast senescence and dysfunction. However, the regulatory mechanism that controls the ROS-induced senescence of osteoblasts is poorly understood. Here, we identified Peptidyl arginine deiminase 2 (PADI2), a post-translational modifying enzyme, as a regulator of ROS-accelerated senescence of osteoblasts via RNA-sequencing and further functional validations. PADI2 downregulation by treatment with H2O2 or its siRNA promoted cellular senescence and suppressed osteoblast differentiation. CCL2, 5, and 7 known as the elements of the senescence-associated secretory phenotype (SASP) which is a secretome including proinflammatory cytokines and chemokines emitted by senescent cells and a representative feature of senescence, were upregulated by H2O2 treatment or Padi2 knockdown. Furthermore, blocking these SASP factors with neutralizing antibodies or siRNAs alleviated the senescence and dysfunction of osteoblasts induced by H2O2 treatment or Padi2 knockdown. The elevated production of these SASP factors was mediated by the activation of NFκB signaling pathway. The inhibition of NFκB using the pharmacological inhibitor or siRNA effectively relieved H2O2 treatment- or Padi2 knockdown-induced senescence and osteoblast dysfunction. Together, our study for the first time uncover the role of PADI2 in ROS-accelerated cellular senescence of osteoblasts and provide new mechanistic and therapeutic insights into excessive ROS-promoted cellular senescence and aging-related bone diseases.

The STAT3 inhibitor Stattic acts independently of STAT3 to decrease histone acetylation and modulate gene expression.

Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor involved in many physiological functions including embryonic development and immune responses and is often activated under pathological conditions such as cancer. Strategies to inactivate STAT3 are being pursued as potential anticancer therapies and have led to the identification of Stattic (6-nitrobenzo[b]thiophene-1,1-dioxide) as a "specific" STAT3 inhibitor that is often used to interrogate STAT3-mediated gene expression in vitro and in vivo. Here, we show that Stattic exerts many STAT3-independent effects on cancer cells, calling for reassessment of results previously ascribed to STAT3 functions. Studies of the STAT3-deficient prostate cancer cell line PC-3 (PC3) along with STAT3-proficient breast cancer cell lines (MDA-MB-231, SUM149) revealed that Stattic attenuated histone acetylation and neutralized effects of the histone deacetylase (HDAC) inhibitor romidepsin. In PC3 cells, Stattic alone inhibited gene expression of CCL20 and CCL2, but activated expression of TNFA, CEBPD, SOX2, and MYC. In addition, we found that Stattic promoted autophagy and caused cell death. These data point to profound epigenetic effects of Stattic that are independent of its function as a STAT3 inhibitor. Our results demonstrate that Stattic directly or indirectly reduces histone acetylation and suggest reevaluation of Stattic and related compounds as polypharmacological agents through multipronged cytotoxic effects on cancer cells.

The molecular structure and role of CCL2 (MCP-1) and C-C chemokine receptor CCR2 in skeletal biology and diseases.

Monocyte chemoattractant protein-1, also called chemokine (C-C motif) ligand 2 (CCL2) or small inducible cytokine A2, is an inflammatory mediator capable of recruiting monocytes, memory T cells, and dendritic cells. CCL2 is a member of the CC chemokine superfamily, which binds to its receptor, C-C motif chemokine receptor-2 (CCR2), for the induction of chemotactic activity and an increase of calcium influx. It exerts multiple effects on a variety of cells, including monocytes, macrophages, osteoclasts, basophils, and endothelial cells, and is involved in a diverse range of diseases. This review discusses the molecular structure and role of CCL2 and CCR2 in skeletal biology and disease. Molecular structure analyses reveal that CCL2 shares a conserved C-C motif; however, it has only limited sequence homology with other CCL family members. Likewise, CCR2, as a member of the G-protein-coupled seven-transmembrane receptor superfamily, shares conserved cysteine residues, but exhibits very limited sequence homology with other CCR family members. In the skeletal system, the expression of CCL2 is regulated by a variety of factors, such as parathyroid hormone/parathyroid hormone-related peptide, interleukin 1b, tumor necrosis factor-α and transforming growth factor-beta, RANKL, and mechanical forces. The interaction of CCL2 and CCR2 activates several signaling cascades, including PI3K/Akt/ERK/NF-κB, PI3K/MAPKs, and JAK/STAT-1/STAT-3. Understanding the role of CCL2 and CCR2 will facilitate the development of novel therapies for skeletal disorders, including rheumatoid arthritis, osteolysis and other inflammatory diseases related to abnormal chemotaxis.

Wolfberry-derived zeaxanthin dipalmitate delays retinal degeneration in a mouse model of retinitis pigmentosa through modulating STAT3, CCL2 and MAPK pathways.

Retinitis pigmentosa (RP) is a group of inherited photoreceptor degeneration diseases that causes blindness without effective treatment. The pathogenesis of retinal degeneration involves mainly oxidative stress and inflammatory responses. Zeaxanthin dipalmitate (ZD), a wolfberry-derived carotenoid, has anti-inflammatory and anti-oxidative stress effects. Here we investigated whether these properties of ZD can delay the retinal degeneration in rd10 mice, a model of RP, and explored its underlying mechanism. One shot of ZD or control vehicle was intravitreally injected into rd10 mice on postnatal day 16 (P16). Retinal function and structure of rd10 mice were assessed at P25, when rods degenerate substantially, using a visual behavior test, multi-electrode-array recordings and immunostaining. Retinal pathogenic gene expression and regulation of signaling pathways by ZD were explored using transcriptome sequencing and western blotting. Our results showed that ZD treatment improved the visual behavior of rd10 mice and delayed the degeneration of retinal photoreceptors. It also improved the light responses of photoreceptors, bipolar cells and retinal ganglion cells. The expression of genes that are involved in inflammation, apoptosis and oxidative stress were up-regulated in rd10 mice, and were reduced by ZD. ZD further reduced the activation of two key factors, signal transducer and activator of transcription 3 and chemokine (C-C motif) ligand 2, down-regulated the expression of the inflammatory factor GFAP, and inhibited extracellular signal regulated protein kinases and P38, but not the JNK pathways. In conclusion, ZD delays the degeneration of the rd10 retina both morphologically and functionally. Its anti-inflammatory function is mediated primarily through the signal transducer and activator of transcription 3, chemokine (C-C motif) ligand 2 and MAPK pathways. Thus, ZD may serve as a potential clinical candidate to treat RP.

Adropin decreases endothelial monolayer permeability after cell-free hemoglobin exposure and reduces MCP-1-induced macrophage transmigration.

BACKGROUND: Cell-free heme-containing proteins mediate endothelial injury in a variety of disease states including subarachnoid hemorrhage and sepsis by increasing endothelial permeability. Inflammatory cells are also attracted to sites of vascular injury by monocyte chemotactic protein 1 (MCP-1) and other chemokines. We have identified a novel peptide hormone, adropin, that protects against hemoglobin-induced endothelial permeability and MCP-1-induced macrophage migration. METHODS: Human microvascular endothelial cells were exposed to cell-free hemoglobin (CFH) with and without adropin treatment before measuring monolayer permeability using a FITC-dextran tracer assay. mRNA and culture media were collected for molecular studies. We also assessed the effect of adropin on macrophage movement across the endothelial monolayer using an MCP-1-induced migration assay. RESULTS: CFH exposure decreases adropin expression and increases paracellular permeability of human endothelial cells. Treating cells with synthetic adropin protects against the increased permeability observed during the natural injury progression. Cell viability was similar in all groups and Hmox1 expression was not affected by adropin treatment. MCP-1 potently induced macrophage migration across the endothelial monolayer and adropin treatment effectively reduced this phenomenon. CONCLUSIONS: Endothelial injury is a hallmark of many disease states. Our results suggest that adropin treatment could be a valuable strategy in preventing heme-mediated endothelial injury and macrophage infiltration. Further investigation of adropin therapy in animal models and human tissue specimens is needed.

Repurposing bortezomib for choroidal neovascularization treatment via antagonizing VEGF-A and PDGF-D mediated signaling.

Neovascular age-related macular degeneration (neoAMD) is the leading cause of blindness in AMD and manifests as choroidal neovascularization (CNV). Anti-vascular endothelial growth factor (VEGF) therapies are the mainstay treatments but with limited efficacy and cause detrimental effects on the retina after long-term application. These disadvantages warrant alternative strategy. Herein, we examined the effect on CNV by intravitreal injection of bortezomib, a reversible proteasome inhibitor, and further dissected the mechanism. Krypton red Laser was used to create CNV model in mice. The angiogenesis volume was assessed in choroidal flat-mount with isolectin GS-IB4 labeling and the leakage was examined with fluorescein fundus angiography. Injection of Borsub inhibited angiogenesis in the CNV model which was dose-dependent; the injection significantly inhibited leakage as well. Furthermore, Borsub injection reduced the contents of VEGF-A, macrophage chemotactic factor 1 (MCP-1), and platelet-derived growth factor (PDGF)-D but not PDGF-B, examined by enzyme-linked immunosorbent assay, in choroid/retinal pigment epithelium (RPE) tissue. These injections also reduced phospho-VEGFR-2 and phospho-PDGFRß in choroid/RPE tissue examined by immunoblotting. Moreover, Borsub inhibited the recruitment of mural cells or macrophages to laser-injured spots. Injection of Borsub indicated negative effect on scotopic and photopic responses recorded by electroretinogram. Altogether, intravitreal injection of Borsub significantly reduced CNV by antagonizing VEGF-A/Flk-1 and PDGF-D/PDGFRß pathways without impacting electroretinography parameters. Thus, Borsub may offer an invaluable therapy for the prevention and treatment of neoAMD.

Effect of a monocyte chemoattractant protein-1 synthesis inhibitor on fibroblasts from patients with carpal tunnel syndrome.

BACKGROUND: Carpal Tunnel Syndrome (CTS) is an idiopathic fibrotic disorder. Fibrosis in the subsynovial connective tissues (SSCT) of CTS and many other fibrotic diseases is mediated by Transforming growth factor ß (TGF-ß). Recently monocyte chemoattractant protein-1 (MCP-1) a cytokine involved in cellular recruitment has been suggested to regulate TGF-ß activity. It is related to the onset of diseases which are caused by fibrosis, such as idiopathic pulmonary fibrosis, renal fibrosis, and systemic scleroderma. In this study, we evaluated the effect of the MCP-1 synthesis inhibitor, Bindarit, on primary cultures of fibroblasts from the SSCT of five CTS patients. METHODS: Fibroblasts were treated with Bindarit (10 µM, 50 µM, 100 µM, or 300 µM). Responses to inhibitors were evaluated by regulation of CTS fibrosis-associated genes, fibrosis gene array and Smad luciferase reporter assay. We also assessed the combination effect of Bindarit and SD208, a TGF-ß receptor type 1 inhibitor on TGF-ß signaling. RESULTS: Collagen type III A1 (Col3), connective tissue growth factor (CTGF), and SERPINE1 expression were significantly down-regulated by Bindarit (300 µM) compared to vehicle control. In the fibrosis array, expression of inhibin beta E chain precursor (INHBE), beta actin (ACTB), endothelin 1 (EDN1) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) were significantly down-regulated, and integrin beta-3 (ITGB3) was significantly up-regulated by Bindarit (300 µM). Smad signal transduction activation was significantly down-regulated by Bindarit (300 µM) and/or SD208 (1 µM) with TGF-ß1 compared to vehicle control with TGF-ß1. CONCLUSIONS: These results suggest that Bindarit in combination with SD208 may be beneficial as medical therapy for the SSCT fibrosis associated with CTS.

Critical roles of astrocytic-CCL2-dependent monocyte infiltration in a DJ-1 knockout mouse model of delayed brain repair.

Monocyte-derived macrophages play a role in the repair of the injured brain. We previously reported that a deficiency of the Parkinson's disease (PD)-associated gene DJ-1 delays repair of brain injury produced by stereotaxic injection of ATP, a component of damage-associated molecular patterns. Here, we show that a DJ-1 deficiency attenuates monocyte infiltration into the damaged brain owing to a decrease in C-C motif chemokine ligand 2 (CCL2) expression in astrocytes. Like DJ-1-knockout (KO) mice, CCL2 receptor (CCR2)-KO mice showed defects in monocyte infiltration and delayed recovery of brain injury, as determined by 9.4 T magnetic resonance imaging analysis and immunostaining for tyrosine hydroxylase and glial fibrillary acid protein. Notably, transcriptome analyses showed that genes related to regeneration and synapse formation were similarly downregulated in injured brains of DJ-1-KO and CCR2-KO mice compared with the injured wild-type brain. These results indicate that defective astrogliosis in DJ-1-KO mice is associated with decreased CCL2 expression and attenuated monocyte infiltration, resulting in delayed repair of brain injury. Thus, delayed repair of brain injury could contribute to the development of PD. MAIN POINTS: A DJ-1 deficiency attenuates infiltration of monocytes owing to a decrease in CCL2 expression in astrocytes, which in turn led to delay in repair of brain injury.

CCL2 mediates early renal leukocyte infiltration during salt-sensitive hypertension.

Studies examining mechanisms of Dahl salt-sensitive (SS) hypertension have implicated the infiltration of leukocytes in the kidneys, which contribute to renal disease and elevated blood pressure. However, the signaling pathways by which leukocytes traffic to the kidneys remain poorly understood. The present study nominated a signaling pathway by analyzing a kidney RNA sequencing data set from SS rats fed either a low-salt (0.4% NaCl) diet or a high-salt (4.0% NaCl) diet. From this analysis, chemokine (C-C motif) ligand 2 (CCL2) and chemokine (C-C motif) receptor 2 (CCR2) were nominated as a potential pathway modifying renal leukocyte infiltration and contributing to SS hypertension. The functional role of the CCL2/CCR2 pathway was tested by daily administration of CCR2 antagonist (RS-102895 at 5 mg·kg-1·day-1 in DMSO) or DMSO vehicle for 3 or 21 days by intraperitoneal injections during the high salt challenge. Blood pressure, renal leukocyte infiltration, and renal damage were evaluated. The results demonstrated that RS-102895 treatment ameliorated renal damage (urinary albumin excretion; 43.4 ± 5.1 vs. 114.7 ± 15.2 mg/day in vehicle, P < 0.001) and hypertension (144.3 ± 2.2 vs. 158.9 ± 4.8 mmHg in vehicle, P < 0.001) after 21 days of high-salt diet. It was determined that renal leukocyte infiltration was blunted by day 3 of the high-salt diet (1.4 ± 0.1 vs. 1.9 ± 0.2 in vehicle × 106 CD45+ cells/kidney, P = 0.034). An in vitro chemotaxis assay validated the effect of RS-102895 on leukocyte chemotaxis toward CCL2. The results suggest that increased CCL2 in SS kidneys is important in the early recruitment of leukocytes, and blockade of this recruitment by administering RS-102895 subsequently blunted the renal damage and hypertension.

Phase I dose-escalation trial to repurpose propagermanium, an oral CCL2 inhibitor, in patients with breast cancer.

The formation of premetastatic niches creates a fertile environment for the seeding of disseminated cancer cells in selected secondary organs. This is crucial for the development of metastasis in various malignancies, including breast cancer (BC). We previously reported that the loss of FBXW7 in bone marrow-derived stromal cells promoted cancer metastasis by increasing the production of the chemokine CCL2, which attracts myeloid-derived suppressor cells and macrophages to the premetastatic niche. Furthermore, treatment with the CCL2 inhibitor propagermanium (PG), which has been used in Japan as a therapeutic agent against chronic hepatitis B, was shown to block the enhancement of metastasis in FBXW7-deficient mice through inhibiting the formation of premetastatic niches. Here, we describe a phase I dose-escalation study of PG used as an antimetastatic drug for perioperative patients with primary BC. The primary end-point was the percentage of patients who experience dose-limiting toxicity. Twelve patients were enrolled in the study. Dose-limiting toxicity was not observed, and the maximum dose was determined to be 90 mg/body/day. The serum concentrations of PG were nearly within the normal range in all observation days. We observed an inverse correlation between FBXW7 mRNA levels in blood and the serum concentrations of CCL2 and interleukin (IL)-6, in agreement with our previous mouse model. Also, IL-6 was downregulated in a PG dose-dependent manner, as observed in mice. Thus, PG was given safely and it is expected to have antimetastatic potential in BC. This trial is registered in the UMIN Clinical Trials Registry as UMIN000022494.

Phase 1b study of a small molecule antagonist of human chemokine (C-C motif) receptor 2 (PF-04136309) in combination with nab-paclitaxel/gemcitabine in first-line treatment of metastatic pancreatic ductal adenocarcinoma.

Background In pancreatic ductal adenocarcinoma (PDAC), the chemokine (C-C motif) ligand 2 (CCL2)/chemokine (C-C motif) receptor 2 (CCR2) axis plays a key role in immunosuppressive properties of the tumor microenvironment, patient prognosis, and chemoresistance. This phase Ib study assessed the effects of the orally administered CCR2 inhibitor PF-04136309 in combination with nab-paclitaxel and gemcitabine in patients with previously untreated metastatic PDAC. Methods Patients received PF-04136309 twice daily (BID) continuously plus nab-paclitaxel (125 mg/m2) and gemcitabine (1000 mg/m2) administered on days 1, 8, and 15 of each 28-day cycle. The primary objectives were to evaluate safety and tolerability, characterize dose-limiting toxicities (DLTs), and determine the recommended phase II dose (RP2D) of PF-04136309. Results In all, 21 patients received PF-04136309 at a starting dose of 500 mg or 750 mg BID. The RP2D was identified to be 500 mg BID. Of 17 patients treated at the 500 mg BID starting dose, three (17.6%) experienced a total of four DLTs, including grade 3 dysesthesia, diarrhea, and hypokalemia and one event of grade 4 hypoxia. Relative to the small number of patients (n = 21), a high incidence (24%) of pulmonary toxicity was observed in this study. The objective response rate for 21 patients was 23.8% (95% confidence interval: 8.2-47.2%). Levels of CD14 + CCR2+ inflammatory monocytes (IM) decreased in the peripheral blood, but did not accumulate in the bone marrow. Conclusions PF-04136309 in combination with nab-paclitaxel plus gemcitabine had a safety profile that raises concern for synergistic pulmonary toxicity and did not show an efficacy signal above nab-paclitaxel and gemcitabine. ClinicalTrials.gov identifier: NCT02732938.

Forsythoside A inhibits adhesion and migration of monocytes to type II alveolar epithelial cells in lipopolysaccharide-induced acute lung injury through upregulating miR-124.

Acute lung injury (ALI) is a severe disease for which effective drugs are still lacking at present. Forsythia suspensa is a traditional Chinese medicine commonly used to relieve respiratory symptoms in China, but its functional mechanisms remain unclear. Therefore, forsythoside A (FA), the active constituent of F. suspensa, was studied in the present study. Inflammation models of type II alveolar epithelial MLE-12 cells and BALB/c mice stimulated by lipopolysaccharide (LPS) were established to explore the effects of FA on ALI and the underlying mechanisms. We found that FA inhibited the production of monocyte chemoattractant protein-1 (MCP-1/CCL2) in LPS-stimulated MLE-12 cells in a dose-dependent manner. Moreover, FA decreased the adhesion and migration of monocytes to MLE-12 cells. Furthermore, miR-124 expression was upregulated after FA treatment. The luciferase report assay showed that miR-124 mimic reduced the activity of CCL2 in MLE-12 cells. However, the inhibitory effects of FA on CCL2 expression and monocyte adhesion and migration to MLE-12 cells were counteracted by treatment with a miR-124 inhibitor. Critically, FA ameliorated LPS-induced pathological damage, decreased the serum levels of tumor necrosis factor-α and interleukin-6, and inhibited CCL2 secretion and macrophage infiltration in lungs in ALI mice. Meanwhile, administration of miR-124 inhibitor attenuated the protective effects of FA. The present study suggests that FA attenuates LPS-induced adhesion and migration of monocytes to type II alveolar epithelial cells though upregulating miR-124, thereby inhibiting the expression of CCL2. These findings indicate that the potential application of FA is promising and that miR-124 mimics could also be used in the treatment of ALI.

Mesenchymal stem/stromal cells stably transduced with an inhibitor of CC chemokine ligand 2 ameliorate bronchopulmonary dysplasia and pulmonary hypertension.

Perinatal bronchopulmonary dysplasia (BPD) is defined as lung injury in preterm infants caused by various factors, resulting in serious respiratory dysfunction and high mortality. The administration of mesenchymal stem/stromal cells (MSCs) to treat/prevent BPD has proven to have certain therapeutic effects. However, MSCs can only weakly regulate macrophage function, which is strongly involved in the development of BPD. 7ND-MSCs are MSCs transfected with 7ND, a truncated version of CC chemokine ligand 2 (CCL2) that promotes macrophage activation, using a lentiviral vector. In the present study, we show in a BPD rat model that 7ND-MSC administration, but not MSCs alone, ameliorated the impaired alveolarization evaluated by volume density and surface area in the lung tissue, as well as pulmonary artery remodeling and pulmonary hypertension induced by BPD. In addition, 7ND-MSCs, but not MSCs alone, reduced M1 macrophages and the messenger RNA expressions of interleukin-6 and CCL2 in the lung tissue. Thus, the present study showed the treatment effect of 7ND-MSCs in a BPD rat model, which was more effective than that of MSCs alone.

Treatment with CCR2 antagonist is neuroprotective but does not alter epileptogenesis in the pilocarpine rat model of epilepsy.

Neuroinflammation role on epileptogenesis has been the subject of increasing interest. Many studies showed elevation in cytokines and chemokines expression following seizures, such as, CCL2 protein (C-C motif ligand 2 chemokine) and its specific receptor, CCR2. In addition, recent studies manipulating the CCL2/CCR2 complex verified improved seizure outcome in different seizure models. In the present study, the effects of CCR2 antagonist was investigated using the pilocarpine rat model of epilepsy. Status epilepticus (SE) was induced by pilocarpine i.p. injection in adult rats. Daily oral treatment with CCR2 antagonist or vehicle was initiated 5 h following SE and lasted 5 or 10 days. Rats were euthanized 5 days after SE to evaluate neuronal damage and glial density or 30 days after SE to investigate spontaneous seizures development and seizure susceptibility to a second hit pentylenetetrazol (PTZ) test. Rats that received CCR2 antagonist presented less degenerating cells at hippocampal CA1 region. There was also a significant decrease in CA1 volume after SE that was not observed in treated rats. On the other hand, microglia cell density increased after SE regardless of CCR2 antagonist use. Treatment with CCR2 antagonist did not alter spontaneous seizure occurrence or later seizure susceptibility to PTZ in chronic rats. Additional rats were pretreated with CCR2 antagonist prior to SE induction, but this did not change SE progression. The data show that oral treatment with CCR2 antagonist is neuroprotective, but does not alter other epileptogenic factors, such as, neuroinflammation, or seizure development, after pilocarpine-induced SE in rats.

(3,3″)-Linked Biflavanones from Ouratea spectabilis and Their Effects on the Release of Proinflammatory Cytokines in THP-1 Cells.

Ouratea spectabilis is an arborous species traditionally used in Brazil as an anti-inflammatory agent. Four new (3,3″)-linked biflavanone O-methyl ethers, named ouratein A (1), B (2), C (3), and D (4), were isolated from the bark extract of the species. Ouratein A (1) is an enantiomer of neochamagesmine A, which has never been described before. The structures were elucidated by extensive spectroscopic data analyses, whereas their absolute configurations were defined by electronic circular dichroism data. Ouratein D (4) inhibited in vitro the release of the pro-inflammatory cytokine CCL2 by lipopolysaccharide-stimulated THP-1 cells (IC50 of 3.1 ± 1.1 µM), whereas TNF and IL-1ß release were not reduced by any of the biflavanones. These findings show ouratein D (4) as a selective CCL2 inhibitor, which may have potential for the development of new anti-inflammatory agents to prevent or treat cardiovascular diseases.

Cessation of CCL2 inhibition accelerates breast cancer metastasis by promoting angiogenesis.

Secretion of C-C chemokine ligand 2 (CCL2) by mammary tumours recruits CCR2-expressing inflammatory monocytes to primary tumours and metastatic sites, and CCL2 neutralization in mice inhibits metastasis by retaining monocytes in the bone marrow. Here we report a paradoxical effect of CCL2 in four syngeneic mouse models of metastatic breast cancer. Surprisingly, interruption of CCL2 inhibition leads to an overshoot of metastases and accelerates death. This is the result of monocyte release from the bone marrow and enhancement of cancer cell mobilization from the primary tumour, as well as blood vessel formation and increased proliferation of metastatic cells in the lungs in an interleukin (IL)-6- and vascular endothelial growth factor (VEGF)-A-dependent manner. Notably, inhibition of CCL2 and IL-6 markedly reduced metastases and increased survival of the animals. CCL2 has been implicated in various neoplasias and adopted as a therapeutic target. However, our results call for caution when considering anti-CCL2 agents as monotherapy in metastatic disease and highlight the tumour microenvironment as a critical determinant of successful anti-metastatic therapy.

Overcoming Drug Resistance to BRAF Inhibitor.

One of the most frequently found mutations in human melanomas is in the B-raf gene, making its protein BRAF a key target for therapy. However, in patients treated with BRAF inhibitor (BRAFi), although the response is very good at first, relapse occurs within 6 months, on the average. In order to overcome this drug resistance to BRAFi, various combinations of BRAFi with other drugs have been explored, and some are being applied clinically, such as a combination of BRAF and MEK inhibitors. Experimental data for melanoma in mice show that under continuous treatment with BRAFi, the pro-cancer MDSCs and chemokine CCL2 initially decrease but eventually increase to above their original level, while the anticancer T cells continuously decrease. In this paper, we develop a mathematical model that explains these experimental results. The model is used to explore the efficacy of combinations of BRAFi with anti-CCL2, anti-PD-1 and anti-CTLA-4, with the aim of eliminating or reducing drug resistance to BRAFi.

Puerarin inhibits the migration of osteoclast precursors and osteoclastogenesis by inhibiting MCP-1 production.

Puerarin inhibits osteoclastogenesis and cells migration. This study aims to explore whether puerarin prevents osteoclastogenesis by inhibiting osteoclast precursors (OCPs) migration. The results showed that puerarin reduced MCP-1 production in OCPs, while inhibiting OCPs migration based on MCP-1. Puerarin reversed MCP-1-promoted osteoclastogenesis. CCR2 overexpression didn't increase osteoclastogenesis with puerarin. Therefore, puerarin prevents OCPs migration by reducing MCP-1, whereby inhibiting osteoclastogenesis.