Monocyte Chemoattractant Protein-1 Gene Expression Research Articles

Effect of simvastatin on monocyte chemoattractant protein-1 expression in endometriosis patients: a randomized controlled trial

BackgroundSimvastatin is a promising new drug for the treatment of endometriosis. It is a cholesterol-lowering drug that acts by inhibiting HMG-CoA reductase, resulting in a decrease in mevalonate, a precursor of cholesterol and monocyte chemoattractant protein-1 (MCP-1). This study investigated the effect of pre-operative oral simvastatin administration on MCP-1 gene expression and serum MCP-1 protein levels in patients with endometriosis.MethodsA prospective, randomized, controlled study was conducted at the Reproductive Endocrinology Unit of the Department of Obstetrics and Gynecology at the Faculty of Medicine Ramathibodi Hospital. Forty women (mean age: 18–45 years) scheduled for laparoscopic surgery who had been diagnosed with endometriosis were recruited and randomly assigned to either a treatment group (20 mg/d of orally administered simvastatin for 2 weeks before surgery) or an untreated control group. Serum was collected before and after treatment and protein levels of MCP-1 were determined. MCP-1 and CD68 transcript levels were also quantified using real-time PCR on endometriotic cyst tissues.ResultsMCP-1 gene expression on endometriotic cyst was not significantly different between the simvastatin-treated and untreated groups (P = 0.99). CD68 expression was higher in the treatment group compared to the control group, but this was not statistically significant (P = 0.055). Serum MCP-1 levels following simvastatin treatment were higher than in samples obtained before treatment (297.89 ± 70.77 and 255.51 ± 63.79 pg/ml, respectively) (P = 0.01).ConclusionsTreatment with 20 mg/d of simvastatin for 2 weeks did not reduce the expression of either the chemokine MCP-1 gene or macrophage-specific genes. Cumulatively, this suggests that simvastatin is not ideal for treating endometriosis because a higher dose of simvastatin (40–100 mg/d) would be needed to achieve the target outcome, which would significantly increase the risk of myopathy in patients.Trial registrationThai Clinical Trials Registry TCTR20130627003 Registered: June 27, 2013.

Inhibition of Monocyte Chemoattractant Protein 1 Prevents Conjunctival Fibrosis in an Experimental Model of Glaucoma Filtration Surgery.

To evaluate the effect of treatment with monocyte chemoattractant protein-1 receptor inhibitor (MCP-Ri) to maintain bleb survival and prevent fibrosis in an experimental model of glaucoma filtration surgery (GFS). GFS was performed on one eye of C57/Bl6 mice (n = 36) that was treated with MCP-Ri, mitomycin-C (MMC), or vehicle at the time of surgery. Real-time polymerase chain reaction was used to evaluate conjunctival expression of monocyte chemoattractant protein-1 (MCP-1), TGFB1, TGFB2, collagen 1a1 (Col1a1), sparc (Sparc), and fibronectin at 2 and 7 days following surgery. Anterior segment slit-lamp examination, optical coherence tomography, and confocal microscopy were performed in vivo at day 14. Eyes were processed for immunohistochemical staining of F4/80, a monocyte-macrophage marker, at day 2. In vitro experiments were also performed to compare the effect of MMC, MCP-Ri, and vehicle on the viability of mouse Tenon's fibroblasts. Treatment with MCP-Ri results in a greater reduction in the percentage of F4/80-positive cells in conjunctival blebs and lesser MCP-1 gene expression following experimental GFS than MMC or control. Both MMC and MCP-Ri reduced Col1a1 and Sparc expression, but not fibronectin. TGFB1 decreased with MCP-Ri but not MMC; MMC but not MCP-Ri reduced TGFB2. MMC and MCP-Ri treatment resulted in the preservation of bleb height at day 14, as compared to control. MCP-Ri was less toxic to mouse Tenon's fibroblasts in comparison with MMC. Targeting MCP-1 results in prolonged bleb survival following experimental GFS with less cellular toxicity as compared to MMC. MCP inhibition could provide a safer alternative to conventional antifibrotic adjunctive treatments in GFS.

A functional SNP MCP-1 (−2518A/G) predispose to renal disorder in Indian Systemic Lupus Erythematosus patients

Systemic Lupus Erythematosus (SLE) is a clinically heterogeneous chronic, inflammatory autoimmune disorder that affects multiple organs where exact etiology of the disease is not yet clearly understood. Various evidences suggest that genetic polymorphisms in inflammatory mediators like cytokines and chemokines may influence development of the disease. Here, we investigated whether functional polymorphism at the Monocyte Chemoattractant Protein-1 (MCP-1) regulatory region associates with disease phenotype in Indian SLE patients. This case control study included 200 SLE patients and 201 ethnically matched healthy controls. Genotyping of MCP-1 (−2518 A/G) polymorphism was performed using PCR-RFLP method. Serum MCP-1 levels were detected by bead-based multiplex immunoassay. Serum MCP-1 levels were found to be higher in patients compared with healthy individuals (p<0.0001). A significant difference for MCP-1G allele frequency (OR=1.9, 95%CI=1.4–2.6, p<0.0001) was observed among SLE patients against healthy individuals. A significant difference in the distribution of MCP-1 −2518GG (OR=3.0, 95%CI=1.4–6.7, p=0.0041) and AG+GG genotypes (OR=2.0, 95%CI=1.4–3.0, p=0.0005) was also noted among SLE patients when compared with healthy individuals. A significant association was observed between A/G and G/G versus A/A genotypes with renal manifestations (p<0.0001, Pc<0.001). Serum MCP-1 levels in active LN patients were found to be significantly higher than inactive LN (p=0.0059), mild LN (p=0.0061) as well as non-LN patients (p=0.0001). These findings suggest that −2518G allele of MCP-1 −2518 A/G polymorphism is associated with renal disorders and may influence MCP-1 gene expression among Indian SLE patients.

RAGE-aptamer Attenuates the Growth and Liver Metastasis of Malignant Melanoma in Nude Mice

Epidemiological studies have suggested a link between cumulative diabetic exposure and cancer. The interaction of advanced glycation end products (AGEs) with their receptor (RAGE) may contribute to the phenomenon. We examined the effects of DNA aptamer raised against RAGE (RAGE-aptamer) on growth and liver metastasis of G361 melanoma in nude mice. Malignant melanoma cells were intradermally injected into the upper flank region of nude mice, which received continuous administration of RAGE-aptamer (38.4 pmol/day/g body weight) or vehicle intraperitoneally by an osmotic pump up to 42 d. RAGE-aptamer significantly reduced levels of 8-hydroxy-2′-deoxy-guanosine, AGEs, RAGE, proliferating nuclear antigen, cyclin D1, vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), and CD31 and Mac-3, respective markers of endothelial cells and macrophages in tumors of nude mice, and suppressed proliferation and liver metastasis of malignant melanoma. Furthermore, RAGE-aptamer attenuated AGE-induced oxidative stress generation, proliferation, and VEGF and MCP-1 gene expression in both G361 melanoma cells and endothelial cells. The present findings suggest that RAGE-aptamer could attenuate melanoma growth and liver metastasis in nude mice by suppressing tumor angiogenesis and macrophage infiltration via inhibition of the AGE-RAGE system. RAGE-aptamer may be a novel therapeutic tool for the treatment of malignant melanoma.

Androgen-androgen receptor system improves chronic inflammatory conditions by suppressing monocyte chemoattractant protein-1 gene expression in adipocytes via transcriptional regulation

Age-related decreases in sex hormones are closely related to chronic inflammation in obesity and metabolic diseases. Particularly, the molecular basis of androgen activity in regulating inflammation and controlling metabolism remains largely unknown. Obese adipocytes secrete monocyte chemoattractant protein-1 (MCP-1), a key chemokine that promotes the infiltration of monocytes/macrophages into adipose tissue, thereby leading to metabolic disorders. Here, we studied the role of androgen-androgen receptor (AR) action in regulating MCP-1 expression in adipose tissue. We observed the induction of Mcp-1 expression in 3T3-L1 adipocytes co-cultured with RAW264.7 macrophages. Additionally, Mcp-1 expression was upregulated by culturing in conditioned medium derived from inflammatory macrophages (M1-Mφ) containing tumor necrosis factor-alpha (TNF-α). We found that sex hormones downregulated TNF-α-induced Mcp-1 and interleukin (Il)-6 expression in 3T3-L1 adipocytes. Furthermore, luciferase-reporter analysis indicated that MCP-1 promoter activity was predominantly suppressed by dihydrotestosterone (DHT)-AR interactions through functional canonical nuclear factor-kappa B (NF-κB) sites, whereas non-canonical NF-κB site containing important flanking sequences exhibited minor contributions to DHT-AR transcriptional repression. These findings suggested that androgen-AR suppressed obesity-induced chronic inflammation in adipose tissue.

Tracking and Therapeutic Value of Human Adipose Tissue-derived Mesenchymal Stem Cell Transplantation in Reducing Venous Neointimal Hyperplasia Associated with Arteriovenous Fistula.

To determine if adventitial transplantation of human adipose tissue-derived mesenchymal stem cells (MSCs) to the outflow vein of B6.Cg-Foxn1(nu)/J mice with arteriovenous fistula (AVF) at the time of creation would reduce monocyte chemoattractant protein-1 (Mcp-1) gene expression and venous neointimal hyperplasia. The second aim was to track transplanted zirconium 89 ((89)Zr)-labeled MSCs serially with positron emission tomography (PET) for 21 days. All animal experiments were performed according to protocols approved by the institutional animal care and use committee. Fifty B6.Cg-Foxn1(nu)/J mice were used to accomplish the study aims. Green fluorescent protein was used to stably label 2.5 × 10(5) MSCs, which were injected into the adventitia of the outflow vein at the time of AVF creation in the MSC group. Eleven mice died after AVF placement. Animals were sacrificed on day 7 after AVF placement for real-time polymerase chain reaction (n = 6 for MSC and control groups) and histomorphometric (n = 6 for MSC and control groups) analyses and on day 21 for histomorphometric analysis only (n = 6 for MSC and control groups). In a separate group of experiments (n = 3), animals with transplanted (89)Zr-labeled MSCs were serially imaged with PET for 3 weeks. Multiple comparisons were performed with two-way analysis of variance, followed by the Student t test with post hoc Bonferroni correction. In vessels with transplanted MSCs compared with control vessels, there was a significant decrease in Mcp-1 gene expression (day 7: mean reduction, 62%; P = .029), with a significant increase in the mean lumen vessel area (day 7: mean increase, 176% [P = .013]; day 21: mean increase, 415% [P = .011]). Moreover, this was accompanied by a significant decrease in Ki-67 index (proliferation on day 7: mean reduction, 81% [P = .0003]; proliferation on day 21: mean reduction, 60%, [P = .016]). Prolonged retention of MSCs at the adventitia was evidenced by serial PET images of (89)Zr-labeled cells. Adventitial transplantation of MSCs decreases Mcp-1 gene expression, accompanied by a reduction in venous neointimal hyperplasia.

Increased MCP-1 gene expression in monocytes of severe OSA patients and under intermittent hypoxia.

Obstructive sleep apnea (OSA) is known to be a risk factor of coronary artery disease. Monocyte chemoattractant protein-1 (MCP-1), as a critical factor for monocyte infiltration, is known to play a role in the development of atherosclerosis. This study aimed to investigate the effect of intermittent hypoxia, the hallmark of OSA, on the MCP-1 expression of monocytes. Peripheral blood was sampled from 61 adults enrolled for suspected OSA. RNA was prepared from the isolated monocytes for the analysis of MCP-1. The effect of in vitro intermittent hypoxia on the regulation and function of MCP-1 was investigated on THP-1 monocytic cells and human monocytes. The mRNA and secreted protein levels were investigated by RT/real-time PCR and enzyme-linked immunosorbent assay, respectively. Monocytic MCP-1 gene expression was found to be increased significantly in severe OSA patients. In vitro intermittent hypoxia was demonstrated to increase the mRNA and protein expression levels of MCP-1 dose- and time-dependently in THP-1 monocytic cells. The MCP-1 mRNA expression in monocytes isolated from OSA patient was induced to a much higher level compared to that from normal control. Pre-treatment with inhibitor for p42/44 MAPK or p38 MAPK suppressed the activation of MCP-1 expression by intermittent hypoxia. This is the first study to demonstrate the increase of MCP-1 gene expression in monocytes of severe OSA patients. In addition, monocytic MCP-1 gene expression can be induced under intermittent hypoxia.

Tofogliflozin, A Highly Selective Inhibitor of SGLT2 Blocks Proinflammatory and Proapoptotic Effects of Glucose Overload on Proximal Tubular Cells Partly by Suppressing Oxidative Stress Generation

Ninety percent of glucose filtered by the glomerulus is reabsorbed by a sodium-glucose cotransporter 2 (SGLT2), which is mainly expressed on S1 and S2 segment of renal proximal tubules. Since SGLT-2-mediated glucose reabsorption is increased under diabetic conditions, selective inhibition of SGLT2 is a potential therapeutic target for the treatment of diabetes. We have recently shown that an inhibitor of SGLT2 has anti-inflammatory and antifibrotic effects on experimental diabetic nephropathy partly by suppressing advanced glycation end products formation and oxidative stress generation in the kidney. However, the direct effects of SGLT2 inhibitor on tubular cell damage remain unclear. In this study, we investigated the effects of tofogliflozin, a highly selective inhibitor of SGLT2 on oxidative stress generation, inflammatory and proapoptotic reactions in cultured human proximal tubular cells exposed to high glucose. Tofogliflozin dose-dependently suppressed glucose entry into tubular cells. High glucose exposure (30 mM) for 4 and 24 h significantly increased oxidative stress generation in tubular cells, which were suppressed by the treatment of tofogliflozin or an antioxidant N-acetylcysteine (NAC). Monocyte chemoattractant protein-1 (MCP-1) gene expression and apoptotic cell death were induced by 4 h- and 8 day-exposure to high glucose, respectively, both of which were also blocked by tofogliflozin or NAC. The present study suggests that SGLT2-mediated glucose entry into tubular cells could stimulate oxidative stress and evoke inflammatory and proapoptotic reactions in this cell type. Blockade of glucose reabsorption in tubular cells by SGLT2 inhibitor might exert beneficial effects on tubulointerstitial damage in diabetic nephropathy.

Puerarin suppresses high glucose-induced MCP-1 expression via modulating histone methylation in cultured endothelial cells.

Hyperglycemia is commonly associated with microcirculation dysfunction. The purpose of the present study was to investigate the epigenetic mechanism involved in the repression of monocyte chemoattractant protein-1 (MCP-1) expression by puerarin under high glucose (25mM) condition. MCP-1 gene expression was measured by Real-Time quantitative Polymerase Chain Reaction (RT-qPCR), the histone 3 lysine 4 methylation (H3K4me) and lysine 9 methylation (H3K9me) were evaluated using chromatin immunoprecipitation assay. Puerarin significantly inhibited high glucose-induced upregulation of H3K4 di- and tri-methylation (H3K4me2/3) on the MCP-1 gene promotor. Additionally, the enrichment of H3K4 histone methyltransferases including MLL, menin and SET7 on the MCP-1 promotor was increased, while the demethylase LSD1 was decreased in EA.hy926 cells following exposure to high glucose. The changes of the above enzymes were reversed by puerarin treatment. The mRNA expression of MCP-1 was increased by LSD1 blockage, while was decreased by MLL3 blockage. Our findings suggested that puerarin plays a critical role in transcriptional repression of high glucose-induced MCP-1 gene expression, at least in part due to alteration of H3K4me2/3 methylation, thus possesses a therapeutic potential in diabetes-induced vascular injuries.

Inhibitory Effect of 3-(4-Hydroxyphenyl)-1-(thiophen-2-yl) prop-2-en-1-one, a Chalcone Derivative on MCP-1 Expression in Macrophages via Inhibition of ROS and Akt Signaling.

Chalcones (1,3-diaryl-2-propen-1-ones), a subfamily of flavonoid, are widely known to possess potent anti-inflammatory and anti-oxidant properties. In this study, we investigated the effect of 3-(4-Hydroxyphenyl)-1-(thio3-(4-Hydroxyphenyl phen-2-yl)prop-2-en-1-one (TI-I-175), a synthetic chalcone derivative, on endotoxin-induced expression of monocyte chemoattractant protein-1 (MCP-1), one of the key chemokines that regulates migration and infiltration of immune cells, and its potential mechanisms. TII-175 potently inhibited MCP-1 mRNA expression stimulated by lipopolysaccharide (LPS) in RAW 264.7 macrophages without significant effect on cell viability. Treatment of cells with TI-I-175 markedly prevented LPS-induced transcriptional activation of activator protein-1 (AP-1) as measured by luciferase reporter assay, while nuclear factor-κB (NF-κB) activity was not inhibited by TI-I-175, implying that TI-I-175 suppressed MCP-1 expression probably via regulation of AP-1. In addition, TI-I-175 treatment significantly inhibited LPS-induced Akt phosphorylation and led to a significant decrease in reactive oxygen species (ROS) production by LPS, which act as up-stream signaling events required for AP-1 activation in RAW 264.7 macrophages. Taken together, these results indicate that TI-I-175 suppresses MCP-1 gene expression in LPS-stimulated RAW 264.7 macrophages via suppression of ROS production and Akt activation.

Role of miR-146a in regulation of the acute inflammatory response to monosodium urate crystals

ObjectivesMicroRNAs (miRNA) are small non-coding RNAs that function as post-transcriptional repressors of gene expression. We hypothesised that miRNA regulate gene expression of proinflammatory cytokines in response to monosodium urate (MSU)...

Involvement of de novo ceramide synthesis in pro-inflammatory adipokine secretion and adipocyte–macrophage interaction

Interaction between adipocytes and macrophages has been suggested to play a central role in the pathogenesis of obesity. Ceramide, a sphingolipid de novo synthesized from palmitate, is known to stimulate pro-inflammatory cytokine secretion from multiple types of cells. To clarify whether de novo synthesized ceramide contributes to cytokine dysregulation in adipocytes and macrophages, we observed cytokine secretion in mature 3T3-L1 adipocytes (L1) and RAW264.7 macrophages (RAW) cultured alone or co-cultured under the suppression of de novo ceramide synthesis.Palmitate enhanced ceramide accumulation and stimulated the expression and secretion of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) in L1. The suppression of serine-palmitoyl transferase, a rate-limiting enzyme of de novo ceramide synthesis, by myriocin or siRNA attenuated those palmitate-induced alterations, and a ceramide synthase inhibitor fumonisin B1 showed similar results. In contrast, the inhibitor of sphingosine kinase or a membrane-permeable ceramide analogue augmented the cytokine secretion. Myriocin effects on the palmitate-induced changes were not abrogated by toll-like receptor-4 blockade. Although palmitate stimulated RAW to secrete tumor necrosis factor-α (TNF-α), it did not significantly increase ceramide content, and neither myriocin nor fumonisin B1 attenuated the TNF-α hypersecretion. The co-culture of L1 with RAW markedly augmented IL-6 and MCP-1 levels in media. Myriocin or fumonisin B1 significantly lowered these cytokine levels and suppressed the gene expression of TNF-α and MCP-1 in RAW and of IL-6 and MCP-1 in L1.In conclusion, de novo synthesized ceramide partially mediates the palmitate effects on pro-inflammatory adipokines and is possibly involved in the interaction with macrophages.

Pulmonary venous hypertension and mechanical strain stimulate monocyte chemoattractant protein-1 release and structural remodelling of the lung in human and rodent chronic heart failure models

IntroductionThe burden of chronic heart failure (HF) is rising owing to an increased survivorship after myocardial infarction (MI). Pulmonary structural remodelling in patients with HF may protect against oedema while...

The role of Iex-1 in the pathogenesis of venous neointimal hyperplasia associated with hemodialysis arteriovenous fistula.

Arteriovenous fistulas (AVFs) used for hemodialysis fail because of venous neointimal hyperplasia (VNH). There are 1,500,000 patients that have end stage renal disease worldwide and the majority requires hemodialysis. In the present study, the role of the intermediate early response gene X-1 (IEX-1), also known as IER-3 in the pathogenesis of VNH was evaluated. In human samples removed from failed AVF, there was a significant increase in IEX-1 expression localized to the adventitia. In Iex-1 −/− mice and wild type (WT) controls, chronic kidney disease was induced and an AVF placed 28 days later by connecting the carotid artery to jugular vein. The outflow vein was removed three days following the creation of the AVF and gene expression analysis demonstrated a significant decrease in vascular endothelial growth factor-A (Vegf-A) and monocyte chemoattractant protein-1 (Mcp-1) gene expression in Iex-1 −/− mice when compared to WT mice (P<0.05). At 28 days after AVF placement, histomorphometric and immune-histochemical analyses of the outflow vein demonstrated a significant decrease in neointimal hyperplasia with an increase in average lumen vessel area associated with a decrease in fibroblast, myofibroblast, and Ly6C staining. There was a decrease in proliferation (Ki-67) and an increase in the TUNEL staining in Iex-1 KO mice compared to WT. In addition, there was a decrease in Vegf-A, Mcp-1, and matrix metalloproteiniase-9 (Mmp-9) staining. Iex-1 expression was reduced in vivo and in vitro using nanoparticles coated with calcitriol, an inhibitor of Iex-1 that demonstrated that Iex-1 reduction results in decrease in Vegf-A. In aggregate, these results indicate that the absence of IEX-1 gene results in reduced VNH accompanied with a decrease in proliferation, reduced fibroblast, myofibroblast, and Ly6C staining accompanied with increased apoptosis mediated through a reduction in Vegf-A/Mcp-1 axis and Mmp-9. Adventitial delivery of nanoparticles coated with calcitriol reduced Iex-1 and VNH.

IgE in the absence of allergen induces the expression of monocyte chemoattractant protein-1 in the rat basophilic cell-line RBL-2H3

Recently, basophils have been suggested to produce inflammatory mediators in response to IgE in the absence of allergens. Monocyte chemoattractant protein-1 (MCP-1) plays an important role in the initiation of inflammatory responses by recruiting various immune cells to the site of allergic inflammation. In the present study, we examined whether IgE under allergen-free conditions could stimulate basophils and lead to the production of MCP-1. Exposure of the rat basophilic cell-line RBL-2H3 to IgE without allergen resulted in a dose- and time-dependent induction of MCP-1 expression at both the mRNA and protein level. Although allergen was not necessary for IgE-induced MCP-1 expression, it was essential for degranulation as determined by β-hexosaminidase release assay. IgE enhanced phosphorylation of MAP kinases including ERK, p38 kinase, and JNK. However, IgE-induced MCP-1 expression was attenuated by inhibitors for JNK and PKC. Concomitantly, IgE induced activation of AP-1, which is an important transcription factor for MCP-1 gene expression in RBL-2H3 cells. Taken together, our results suggest that IgE alone is sufficient to stimulate basophils to increase expression of MCP-1, which in turn might contribute to the inflammatory response.

3T3-L1 Preadipocytes Exhibit Heightened Monocyte-Chemoattractant Protein-1 Response to Acute Fatty Acid Exposure

Preadipocytes contribute to the inflammatory responses within adipose tissue. Whilst fatty acids are known to elicit an inflammatory response within adipose tissue, the relative contribution of preadipocytes and mature adipocytes to this is yet to be determined. We aimed to examine the actions of common dietary fatty acids on the acute inflammatory and adipokine response in 3T3-L1 preadipocytes and differentiated mature adipocytes. Gene expression levels of key adipokines in 3T3-L1 preadipocytes and adipocytes were determined following incubation with palmitic acid, myristic acid or oleic acid and positive inflammatory control, lipopolysaccharide for 2 and 4 h. Inflammatory kinase signalling was assessed by analysis of nuclear factor-κB, p38-mitogen-activated protein kinase and c-jun amino-terminal kinase phosphorylation. Under basal conditions, intracellular monocyte chemoattractant protein-1 and interleukin-6 gene expression levels were increased in preadipocytes, whereas mature adipocytes expressed increased gene expression levels of leptin and adiponectin. Fatty acid exposure at 2 and 4 h increased both monocyte chemoattractant protein-1 and interleukin-6 gene expression levels in preadipocytes to greater levels than in mature adipocytes. There was an accompanying increase of inhibitor of κB-α degradation and nuclear factor-κB (p65) (Ser536) phosphorylation with fatty acid exposure in the preadipocytes only. The current study points to preadipocytes rather than the adipocytes as the contributors to both immune cell recruitment and inflammatory adipokine secretion with acute increases in fatty acids.

Deregulation of inflammatory response in the diabetic condition is associated with increased ischemic brain injury.

BackgroundAlthough elicited inflammation contributes to tissue injury, a certain level of inflammation is necessary for subsequent tissue repair/remodeling. Diabetes, a chronic low-grade inflammatory state, is a predisposing risk factor for stroke. The condition is associated with delayed wound healing, presumably due to disrupted inflammatory responses. With inclusion of the diabetic condition in an experimental animal model of stroke, this study investigates whether the condition alters inflammatory response and influences stroke-induced brain injury.MethodsC57BL/6 mice were fed a diabetic diet (DD) for 8 weeks to induce an experimental diabetic condition or a normal diet (ND) for the same duration. Gene expression of inflammatory factors including monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), CCR2, and CD36 was assessed in the peripheral immune cells and brains of normal and diabetic mice before and after focal cerebral ischemia. The expression of these factors was also determined in lipopolysaccharide (LPS)-treated cultured normal and diabetic macrophages. Ischemic outcome was assessed in these mice at 3 days post-ischemia.ResultsDD intervention in mice resulted in obesity and elevated insulin and glucose level in the blood. The peritoneal immune cells from the diabetic mice showed higher MCP-1 mRNA levels before and after stroke. Compared to normal mice, diabetic mice showed reduced MCP-1, IL-6, and CCR2 gene expression in the brain at 6 h post-ischemia. LPS-stimulated inflammatory responses were also reduced in the diabetic macrophages. The diabetic mice showed larger infarct size and percent swelling.ConclusionsThese results showed that diabetic conditions deregulate acute inflammatory response and that the condition is associated with increased stroke-induced injury. The study suggests that interventions aimed at restoring appropriate inflammatory response in peripheral immune cells/macrophages may be beneficial in reducing stroke-induced brain injury in subjects with chronic inflammatory conditions.

Advanced glycation end products potentiate citrated plasma-evoked oxidative and inflammatory reactions in endothelial cells by up-regulating protease-activated receptor-1 expression

Advanced glycation end products (AGEs) and receptor RAGE interaction contribute to endothelial cell damage in diabetes. Several thrombogenic abnormalities are also involved in diabetic vascular complications. However, the pathological role of thrombin and protease-activated receptor-1 (PAR-1) system in AGE-induced endothelial cell (EC) damage remains unclear. In this study, we investigated the effects of rivaroxaban, an inhibitor of factor Xa on 3% citrated human plasma-evoked reactive oxygen species (ROS) generation and RAGE, monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) gene expression in AGE-exposed ECs. We further examined whether FR171113, an inhibitor of PAR-1 blocked the plasma-induced EC damage and if AGEs increased PAR-1 expression in ECs. Human citrated plasma stimulated ROS generation and RAGE, MCP-1 and ICAM-1 expression in ECs, all of which were potentiated by the treatment with AGEs. Rivaroxaban or FR171113 significantly inhibited these derangements in plasma- or plasma plus AGE-exposed ECs. Moreover, AGEs significantly increased the PAR-1 levels in ECs. The present study suggests that citrated plasma could induce oxidative and inflammatory reactions in ECs via the activation of thrombin-PAR-1 system and that AGEs could potentiate the plasma-evoked EC damages via up-regulation of PAR-1. Blockade of the crosstalk between AGE-RAGE axis and coagulation system by rivaroxaban might be a novel therapeutic target for thromboembolic disorders in diabetes.

Effects of LPS and dietary free fatty acids on MCP-1 in 3T3-L1 adipocytes and macrophages in vitro

Background:High levels of free fatty acids (FFA) have been suggested to be one of the underlying mechanisms for adipose tissue (AT) inflammation and dysfunction in obesity. Human AT produces several adipokines including monocyte chemoattractant protein-1 (MCP-1), which are involved in the pathogenesis of obesity-mediated inflammation.Objective:In this study, we investigated the effects of lipopolysaccharide (LPS) and a panel of dietary FFA on MCP-1 gene and protein expression in adipocytes and macrophages. Furthermore, we investigated whether the effect of LPS and FFA were mediated through the toll-like receptor 4 (TLR4).Methods:3T3-L1 adipocytes and THP-1 macrophages were incubated for 24 h with the following FFA: monounsaturated fatty acid (oleic acid), saturated fatty acid (palmitic acid) and trans fatty acid (elaidic acid; 500 μM) with and without LPS (2 ng ml−1), and MCP-1 and TLR4 mRNA expression and MCP-1 protein secretion was determined.Results:The results showed that LPS significantly increased MCP-1 and TLR4 expression and MCP-1 secretion in 3T3-L1 adipocytes, and that the MCP-1 expression was blocked by a TLR4 inhibitor (CLI095). The effects of the various FFA on MCP-1 mRNA expression and protein secretion in the adipocytes showed no significant changes either alone or in combination with LPS. In macrophages, palmitic acid increased MCP-1 mRNA expression by 1.8-fold (P<0.05), but oleic acid and elaidic acid had no effects.Conclusions:In conclusion, in 3T3-L1 adipocyte, the TLR4-agonist, LPS, stimulates the proinflammatory chemokine MCP-1. The different classes of FFA did not induce MCP-1 mRNA expression or protein secretion in the adipocytes, but the saturated FFA, palmitic acid, induced MCP-1 mRNA expression in macrophages, possibly because of the higher expression level of TLR4 in the macrophages than the adipocytes. Our results indicate that FFA may induce AT inflammation through proinflammatory stimulation of macrophages.

Pigment epithelium-derived factor (PEDF) binds to caveolin-1 and inhibits the pro-inflammatory effects of caveolin-1 in endothelial cells

Pigment epithelium-derived factor (PEDF) exerts atheroprotective effects both in cell culture and animal models through its anti-oxidative and anti-inflammatory properties. Caveolin-1 (Cav), a major protein component of caveolae in endothelial cells (ECs), plays a role in the progression of atherosclerosis. However, effects of PEDF on Cav-exposed ECs remain unknown. In this study, we examined whether and how PEDF could inhibit the Cav-induced inflammatory and thrombogenic reactions in human umbilical vein ECs (HUVECs). Surface plasmon resonance revealed that PEDF bound to Cav at the dissociation constant of 7.36×10−7M. Further, one of the major Cav-interacting proteins in human serum was identified as PEDF by peptide mass fingerprinting analysis using BIAcore 1000 combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Exogenously added Cav was taken up into the membrane fraction of HUVECs and dose-dependently increased monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1) and plasminogen activator inhibitor-1 (PAI-1) mRNA levels, all of which were blocked by the simultaneous treatment with 10nM PEDF. Small interfering RNAs directed against Cav decreased endogenous Cav levels and suppressed gene expression of MCP-1, VCAM-1 and PAI-1 in HUVECs. This study indicates that PEDF binds to Cav and could block the inflammatory and thrombogenic reactions in Cav-exposed HUVECs. Our present study suggests that atheroprotective effects of PEDF might be partly ascribed to its Cav-interacting properties.