MCP-1 Gene Research Articles

Abstract 262: Circulating Exosomes from PAD Patients Modulate Vascular Repair and Inflammation

Objectives: Peripheral arterial disease (PAD) is a chronic disease characterized by inflammation. Recent work suggests that circulating exosomes may contribute to vascular injury and remodeling. We hypothesize that exosomes from PAD subjects negatively modulate vascular repair via miRNA and bioactive lipid mediators (LM). Methods: Exosomes (particle size 30-100nm) were isolated from plasma of healthy (n=6) and PAD (n=6) subjects. Exosome miRNA was isolated and assessed by qPCR. Targeted metabolo-lipidomics was performed by liquid-chromatography-tandem mass spectrometry. VSMC and EC migration were assessed via scratch assay. Monocyte-derived macrophage gene expression after exposure to exosomes was assessed via RT-qPCR. Results: Compared to healthy subjects, exosomes from PAD subjects contained lower levels of pro-angiogenic miR-126 and miR-210 (25.2±6.4 vs 8.3±1.7, p<0.05 and 0.29±0.07 vs 0.08±0.02, p<0.05, respectively). Exosomes contained arachidonic acid, eicosapentaenoic acid and docosapentanoic acid, as well as both pro-inflammatory and pro-resolving bioactive LMs and their pathway markers, including prostaglandins, leukotrienes, lipoxins, resolvins (D- and E-series) and maresins. By principle component analysis, exosome LM profiles differed significantly between healthy and PAD subjects. Exosomes from PAD subjects increased VSMC migration (1.5±.09-fold vs 1.0±.09-fold wound closure, p<0.005) and decreased EC migration (1.5±.04-fold vs. 1.8±.06-fold wound closure, p<0.005) compared to healthy controls. Both PAD and healthy exosomes increased MDM expression of pro-inflammatory genes TNF-α and MCP-1. Conclusion: Plasma-derived exosomes from PAD patients contain an altered profile of vascular-active miRNA and LMs and confer effects on VSMCs and ECs that may impair vessel remodeling. We describe the first known evidence that plasma exosomes contain pro-resolving LMs. Collectively these data suggest that circulating exosome-based signaling may modulate vascular inflammation and repair in PAD patients.

Prolactin protects against cytokine-induced beta-cell death by NFκB and JNK inhibition.

Type 1 diabetes is caused by an autoimmune assault that induces progressive beta-cell dysfunction and dead. Pro-inflammatory cytokines, such as interleukin 1 beta (IL1B), tumor necrosis factor (TNF) and interferon gamma (IFNG) contribute for beta-cell death, which involves the activation of the nuclear factor kappa B (NFκB) and c- Jun N-terminal kinase (JNK). Prolactin (PRL), a physiological mediator for beta-cell proliferation, was shown to protect beta cells against cytokines pro-apoptotic effects. We presently investigated the mechanisms involved in the protective effects of prolactin against cytokine-induced beta-cell death. The findings obtained indicate that STAT3 activation is involved in the anti-apoptotic role of PRL in rat beta cells. PRL prevents the activation of JNK via AKT and promotes a shift from expression of pro- to anti-apoptotic proteins downstream of the JNK cascade. Furthermore, PRL partially prevents the activation of NFκB and the transcription of its target genes IkBa, Fas, Mcp1, A20 and Cxcl10 and also decreases NO production. On the other hand, the pro-survival effects of PRL do not involve modulation of cytokine-induced endoplasmic reticulum stress. These results suggest that the beneficial effects of PRL in beta cells involve augmentation of anti-apoptotic mechanisms and, at the same time, reduction of pro-apoptotic effectors, rendering beta cells better prepared to deal with inflammatory insults. The better understanding of the pro-survival mechanisms modulated by PRL in beta cells can provide tools to prevent cell demise during an autoimmune attack or following islet transplantation.

Functional blocking of Ninjurin1 as a strategy for protecting endothelial cells in diabetes mellitus.

Ongoing efforts to remove pathological inflammatory stimuli are crucial for the protection of endothelial cells in diabetes. Nerve injury-induced protein 1 (Ninj1) is an adhesion molecule that not only contributes to inflammation but also regulates the apoptosis of endothelial cells. In the present study, Ninj1 was found highly expressed in endothelial cells in Type 2 diabetic mice and increased in high-glucose (HG) cultured HUVECs. Furthermore, we found that Ninj1 levels are up-regulated in endothelial cells in clinical specimens of diabetic patients when compared with nondiabetic tissues, indicating a biological correlation between Ninj1 and endothelial pathophysiology in diabetic condition. Functional blocking of Ninj1 promoted endothelial tube formation and eNOS phosphorylation in the HG condition. Additionally, blocking Ninj1 inhibited the activation of caspase-3 and increased the Bcl-2/Bax ratio, thus inhibiting HUVECs apoptosis induced by HG. HG-induced ROS overproduction, p38 MAPK and NF-κB activation, and the overexpression of VCAM-1, ICAM-1, MCP-1, and IL-6 genes were ameliorated after Ninj1 was blocked. Using the signaling pathway inhibitor LY294002, we found that Bcl-2 expression and eNOS phosphorylation after Ninj1 blockade were regulated via PI3K/Akt signaling pathway. The in vivo endothelial contents, α-SMA+PECAM-1+ vascular numbers, and blood perfusion in the hindlimb were markedly up-regulated after Ninj1 was blocked. According to our findings, functional blocking of Ninj1 shows protective effects on diabetic endothelial cells both in vitro and in vivo Thus, we consider Ninj1 to be a potential therapeutic target for preventing endothelial dysfunction in diabetes mellitus.

Monocyte Chemoattractant Protein-1 in Antineutrophil Cytoplasmic Autoantibody-Associated Vasculitis: Biomarker Potential and Association with Polymorphisms in the MCP-1 and the CC Chemokine Receptor-2 Gene.

Antineutrophil cytoplasmic autoantibody- (ANCA-) associated vasculitis (AAV) are relapsing-remitting disorders with unpredictable prognosis. There is a need of biomarkers for distinguishing which patients will have a more severe outcome and also for predicting relapses in disease activity. This study confirms the previous results of urinary MCP-1 (uMCP-1) as a prognostic marker and explores its potential as a marker of disease activity. Method. 114 patients with AAV were followed regularly between 2002 and 2011 at Skåne University Hospital. Urine samples, blood samples, and clinical status were registered. The urine samples were analyzed in an in-house-developed ELISA. PCR-RLFP was used to analyze the MCP-1 and CCR2 genes. Results. Patients with severe prognosis had significantly higher levels of uMCP-1 compared to patients with nonsevere prognosis and healthy controls. Patients with renal damage had higher levels compared to patients who did not have renal damage. There was also a tendency of higher uMCP-1 levels in active disease as compared to remission. AA in the -2518 position in the MCP-1 gene was associated with a more severe outcome compared to the A/G or the G/G genotype. The A/A genotype were also associated with higher levels of uMCP-1. No significant associations were seen for the CCR2-V64I. Conclusion. This study confirmed the connection between high uMCP-1 levels and poor prognosis and also disease activity. It also suggests an association of the A/A genotype at position -2518 in the MCP-1 gene and poor prognosis in AAV. uMCP-1 is clearly a candidate biomarker of potential clinical value. The A/A genotype association needs further evaluation.

Display of human and rabbit monocyte chemoattractant protein-1 on human embryonic kidney 293T cell surface.

Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a protein that is secreted immediately upon endothelial injury, and thereby it plays a key role in inflammation via recruitment of leucocytes to the site of inflammation at the beginning and throughout the inflammatory processes. Aim of this study was to develop two separate cell lines displaying either human MCP-1 (HMCP-1) or rabbit MCP-1 (RMCP-1) on their surface. A DNA fragment containing HMCP-1- or RMCP-1-encoding sequence was inserted into a pcDNA plasmid. Escherichia coli cells strain TOP 10F’ was separately transformed with the pcDNA/RMCP-1 or /HMCP-1 ligation mixture. Following the cloning and construct verification, human embryonic kidney cell line (HEK 293T) was transfected with either of the linearized plasmids. Plasmid integration into the genomic DNA of HEK 293T cells was verified by polymerase chain reaction (PCR). HMCP-1 and RMCP-1 expression was evaluated at RNA and protein levels by real-time PCR and flow cytometry, respectively. PCR products of the expected sizes were amplified from the chromosomal DNA of transfected HEK 293T cells, i.e. 644 bp for H-MCP1 and 737 bp for RMCP-1 constructs. Real-time PCR revealed that the copy numbers of RMCP1 and HMCP1 mRNA per cell were 294 and 500, respectively. Flow cytometry analysis indicated 85% for RMCP-1 and 87% for HMCP-1 expression levels on the surface of transfected cells, when compared with an isotype control. The experiments thus confirmed that the MCP-1 genes were integrated into the HEK 293T genomic DNA and the encoded proteins were stably expressed on the cell surface.

Evolution of Periodontal Disease: Immune Response and RANK/RANKL/OPG System.

The aim of this study was to evaluate markers of bone loss and immune response present in evolution of periodontal disease. One hundred and two Wistar rats were divided into three animals groups: PD0, without ligation and PD15 days and PD60 days, submitted to ligation placement with a sterile 3-0 silk cord in the cervical region of the upper first molar on both sides. Samples were obtained from the gingival tissue for histomorphometric analysis, immunohistochemical analysis of RANK, RANKL, OPG, characterization of the inflammatory infiltrate, quantification of nitric oxide, MCP-1, RANTES, IP10 chemokines, and expression of the TGF-b1, VEG, and bFGF. The number of inflammatory cells in gingival tissue was higher in PD60 samples. The collagen content and the area occupied by birefringent collagen fibers were lower for PD60. Differential leukocyte counting showed that there was a significantly higher polymorphonuclear influx in group PD15, while PD60 showed a greater number of lymphocytes. PD60 showed higher RANTES, IP-10, MCP-1 gene transcripts, as well as a higher nitric oxide concentration. Clinical evaluation revealed that the PD60 group presented an increase in furcal area. In conclusion, in this animal model the increase of RANK/RANKL and HGF markers is related to a specific immune response, and probably contributed to the evolution of periodontal disease. Investigating the effect of these biomarkers can help in targeted therapy for bone resorption, since blocking these can inhibit bone loss.

Determining Candidate Single Nucleotide Polymorphisms in Acquired Laryngotracheal Stenosis.

Despite wide adoption of strategies to prevent injury from prolonged intubation and tracheotomy, acquired laryngotracheal stenosis (ALTS) has not disappeared. ALTS' persistence may be due to patient factors that confer unique susceptibility for some. We sought to identify genetic markers in genes associated with wound healing that could be associated with ALTS. Case-control study. One hundred thirty-eight patients were recruited, 53 patients with ALTS and 85 control patients who underwent intubation or tracheotomy without evidence of ALTS. The patients' DNA was isolated from whole blood. Custom primers were designed, and the TaqMan assay employing allele-specific polymerase chain reaction was used to interrogate single nucleotide polymorphisms (SNPs) rs1799750, rs522616, rs2276109, rs2569190, rs1800469, and rs1024611 of candidate wound healing genes MMP1, MMP3, MMP12, CD14, TGFβ1, and MCP1, respectively. A logistic regression model was used to examine the association of candidate gene polymorphisms with the presence or absence of ALTS. All 138 patients were successfully genotyped. No significant association was found between candidate SNPs and development of ALTS in the overall group. However, subgroup analysis within each ethnicity identified SNPs that are associated with ALTS depending upon the ethnic background. Patient factors such as variations in wound healing due to functional SNPs may shed light on the development of ALTS. There may be a difference in susceptibility to developing ALTS in different ethnic backgrounds. These preliminary findings need to be corroborated in larger population studies. 3b. Laryngoscope, 128:E111-E116, 2018.

An association of the MCP-1 and CCR2 single nucleotide polymorphisms with colorectal cancer prevalence.

We evaluated the connection between the presence of the -2518 A/G MCP-1 as well as 190 G/A CCR2 polymorphic variants and colorectal cancer (CRC) occurrence. Study group consisted of subjects with different stages of CRC as well as healthy controls. Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. W observed an association between the colorectal cancer and the GG genotype of the -2518 A/G MCP-1 single nucleotide polymorphism. No statistically significant correlation was found between CRC and the 190 G/A CCR2 polymorphism. The results of this study support the hypothesis that polymorphism in the MCP-1 gene may contribute to the etiology of colorectal cancer.

Thiamine deficiency activates hypoxia inducible factor-1α to facilitate pro-apoptotic responses in mouse primary astrocytes.

Thiamine is an essential enzyme cofactor required for proper metabolic function and maintenance of metabolism and energy production in the brain. In developed countries, thiamine deficiency (TD) is most often manifested following chronic alcohol consumption leading to impaired mitochondrial function, oxidative stress, inflammation and excitotoxicity. These biochemical lesions result in apoptotic cell death in both neurons and astrocytes. Comparable histological injuries in patients with hypoxia/ischemia and TD have been described in the thalamus and mammillary bodies, suggesting a congruency between the cellular responses to these stresses. Consistent with hypoxia/ischemia, TD stabilizes and activates Hypoxia Inducible Factor-1α (HIF-1α) under physiological oxygen levels. However, the role of TD-induced HIF-1α in neurological injury is currently unknown. Using Western blot analysis and RT-PCR, we have demonstrated that TD induces HIF-1α expression and activity in primary mouse astrocytes. We observed a time-dependent increase in mRNA and protein expression of the pro-apoptotic and pro-inflammatory HIF-1α target genes MCP1, BNIP3, Nix and Noxa during TD. We also observed apoptotic cell death in TD as demonstrated by PI/Annexin V staining, TUNEL assay, and Cell Death ELISA. Pharmacological inhibition of HIF-1α activity using YC1 and thiamine repletion both reduced expression of pro-apoptotic HIF-1α target genes and apoptotic cell death in TD. These results demonstrate that induction of HIF-1α mediated transcriptional up-regulation of pro-apoptotic/inflammatory signaling contributes to astrocyte cell death during thiamine deficiency.

Relationship between MCP1 (-2518A>G) gene variants and ovarian cancer in Turkish population.

The monocyte chemoattractant protein-1 (MCP-1) gene polymorphism(-2518A>G) in the regulatory region of the MCP-1 protein has been reported to be associated with cancer risk. In this study we aimed to investigate the relationship of MCP-1 (-2518A>G) gene polymorphism and ovarian cancer. MCP-1 genotyping was performed using polymerase chain reaction from blood samples ofovarian cancer patient (n=56) and a control groups (n=52).There was a significant difference in MCP1 (-2518A>G) genotypes between the patient and control groups (p=0.049; x2=6.042). AA carriers were significantly higher in the control group (p=0.014) whereas AG genotype and G allele carriers were significantly higher in the ovarian cancer group (p=0.029, p=0.014, respectively). This study suggests that MCP-1 (-2518A>G) AG genotype and G allele could be considered as risk factor for susceptibility to ovarian cancer.

The gut bacterium and pathobiont Bacteroides vulgatus activates NF-κB in a human gut epithelial cell line in a strain and growth phase dependent manner

The gut microbiota is increasingly implicated in the pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC) although the identity of the bacteria that underpin these diseases has remained elusive. The pathobiont Bacteroides vulgatus has been associated with both diseases although relatively little is known about how its growth and functional activity might drive the host inflammatory response. We identified an ATP Binding Cassette (ABC) export system and lipoprotein in B. vulgatus ATCC 8482 and B. vulgatus PC510 that displayed significant sequence similarity to an NF-κB immunomodulatory regulon previously identified on a CD-derived metagenomic fosmid clone. Interestingly, the ABC export system was specifically enriched in CD subjects suggesting that it may be important for colonization and persistence in the CD gut environment. Both B. vulgatus ATCC 8482 and PC510 activated NF-κB in a strain and growth phase specific manner in a HT-29/kb-seap-25 enterocyte like cell line. B. vulgatus ATCC 8482 also activated NF-κB in a Caco-2-NF-κBluc enterocyte like and an LS174T-NF-κBluc goblet cell like cell lines, and induced NF-κB-p65 subunit nuclear translocation and IL-6, IL-8, CXCL-10 and MCP-1 gene expression. Despite this, NF-κB activation was not coincident with maximal expression of the ABC exporter or lipoprotein in B. vulgatus PC510 suggesting that the regulon may be necessary but not sufficient for the immunomodulatory effects.

Frequency of MCP-1 (rs1024611) and CCR2 (rs1799864) gene polymorphisms and its effect on gene expression level in patients with AgP

ObjectiveTo identify the genetic risk markers of aggressive periodontitis (AgP), researchers focus on genetic components that regulate the immune response. Therefore the purpose of this study was to investigate genetic impact of monocyte chemoattractant protein (MCP)-1–2518A/G and CC chemokine receptor 2 (CCR2) −190G/A gene polymorphisms on AgP susceptibility and the effect of this polymorphism on MCP-1 gene expression in patients with AgP. Material and methodsA total of 215 subjects, 108 AgP and 107 periodontally healthy (H) were recruited in this cross-sectional study (NCT02817568). Gene polymorphisms of MCP-1–2518A/G and CCR2–190G/A were analyzed by a standard polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. MCP-1 messenger (m) RNA expression was measured using quantitative real-time (RT)-PCR in peripheral blood leukocytes from 26 AgP and 16H controls. Threshold cycles (Ct) values were obtained from the RT-PCR analysis based on SYBR Green detection and data was normalized via ΔCt. ResultsThere were no differences between AgP and H groups with regard to MCP-1 and CCR2 genotype distribution and allele frequencies (p>0.05). In contrast, the MCP-1 mRNA expression levels were higher in homozygous “AA” control subjects than having G+ genotype and AA homozygous AgP patients. ConclusionsIt can be concluded that MCP-1 and CCR2 polymorphisms are not associated with AgP in Turkish population. Although in AgP patients, there was AA genotype with MCP-1 mRNA expression it can be speculated that gene expression levels in peripheral blood may not reflect the cytokine/chemokine levels of local tissues.

Functional polymorphism rs1024611 in the MCP1 gene is associated with the risk of varicose veins of lower extremities.

Monocyte chemoattractant protein 1 (MCP-1) is a chemokine responsible for monocyte, basophil, and T-lymphocyte attraction. Polymorphism rs1024611 located in the regulatory region of the MCP1 gene has previously been shown to be associated with increased MCP-1 production. In our study, we aimed to examine the association of rs1024611 with the risk of primary varicose veins (PVVs) of lower extremities. The case group comprised 470 patients with PVVs, and the control group included 269 individuals without a history of chronic venous disease. All cases and controls were ethnic Russians. Genotypes were determined by real-time polymerase chain reaction allelic discrimination. Association was studied by logistic regression analysis. We revealed the association of genotype G/G with the increased risk of PVVs (odds ratio [OR], 1.87; 95% confidence interval [CI], 1.02-3.44; P= .04). In the subgroup analysis, association was revealed only in patients with C2 Clinical, Etiology, Anatomy, and Pathophysiology class (allele G: OR, 1.62 [95% CI, 1.13-2.33; P= .008]; genotype G/G: OR, 3.22 [95% CI, 1.43-7.27; P= .005]), in patients with age at onset of PVVs before 30 years (allele G: OR, 1.41 [95% CI, 1.08-1.85; P= .01]; genotype G/G: OR, 2.35 [95% CI, 1.22-4.55; P= .01]), and in patients who declared no family history (allele G: OR, 1.46 [95% CI, 1.02-2.09; P= .04]; genotype G/G: OR, 2.50 [95% CI, 1.11-5.63; P= .03]). Our results provide evidence for MCP-1 involvement in the development of PVVs and indicate thatinflammation could be implicated in the pathogenesis of this condition.

Phloretin Prevents High-Fat Diet-Induced Obesity and Improves Metabolic Homeostasis.

Reactive oxygen species generated as a by-product in metabolism play a central role in the development of obesity and obesity-related metabolic complications. The objective of the current study is to explore the possibility to block obesity and improve metabolic homeostasis via phloretin, a natural antioxidant product from apple tree leaves and Manchurian apricot. Both preventive and therapeutic activities of phloretin were assessed using a high-fat diet-induced obesity mouse model. Phloretin was injected intraperitoneally twice weekly into regular and obese mice fed a high-fat diet. The effects of phloretin treatment on body weight and composition, fat content in the liver, glucose and lipid metabolism, and insulin resistance were monitored and compared to the control animals. Phloretin treatment significantly blocks high-fat diet-induced weight gain but did not induce weight loss in obese animals. Phloretin improved glucose homeostasis and insulin sensitivity and alleviated hepatic lipid accumulation. RT-PCR analysis showed that phloretin treatment suppresses expression of macrophage markers (F4/80 and Cd68) and pro-inflammatory genes (Mcp-1 and Ccr2) and enhances adiponectin gene expression in white adipose tissue. In addition, phloretin treatment elevated the expression of fatty acid oxidation genes such as carnitine palmitoyltransferase 1a and 1b (Cpt1a and Cpt1b) and reduced expression of monocyte chemoattractant protein-1 (Mcp-1), de novo lipogenesis transcriptional factor peroxisome proliferator-activated receptor-γ 2 (Pparγ2), and its target monoacylglycerol O-acyltransferase (Mgat-1) genes. These results provide direct evidence to support a possible use of phloretin for mitigation of obesity and maintenance of metabolic homeostasis.

Pectin from transgenic flax shives regulates extracellular matrix remodelling in human skin fibroblasts

Abstract Pectin, a polysaccharide polymer from the plant cell wall, is an underestimated natural resource with many potential applications in the food and medical industries. Here we present, for the first time, the chemical composition of pectin obtained from flax shives, a by-product of flax fibre processing. The shives from transgenic flax overexpressing β-glucanase were analysed, revealing that genetic modification caused an increase in content of lignin, hemicellulose and pectin, without changes to cellulose, rearrangement of the structure of pectin and cellulose, a decrease in the content of phenolic compounds associated with the cell wall, and an increase in antioxidant capacity of the pectin CDTA fraction. The influence of pectin extract on the extracellular matrix remodelling process was verified. In fibroblast skin cells with induced oxidative stress, addition of pectin caused a reversal of the decrease of mRNA collagen genes, an increase of matrix metalloproteinase, interleukin 6 and MCP-1 gene expression, and a reduction in levels of TIMP-1 and SOCS-1 mRNA. The obtained results, in particular strong antioxidant properties of flax shives pectin from the CDTA-soluble fraction and its significant influence on genes participating in extracellular matrix remodelling, suggest the possible application of flax shives pectin in the wound healing process.

NR4A Receptors Differentially Regulate NF-κB Signaling in Myeloid Cells.

Dysregulation of inflammatory responses is a hallmark of multiple diseases such as atherosclerosis and rheumatoid arthritis. As constitutively active transcription factors, NR4A nuclear receptors function to control the magnitude of inflammatory responses and in chronic inflammatory disease can be protective or pathogenic. Within this study, we demonstrate that TLR4 stimulation using the endotoxin lipopolysaccharide (LPS) rapidly enhances NR4A1–3 expression in human and murine, primary and immortalized myeloid cells with concomitant gene transcription and protein secretion of MIP-3α, a central chemokine implicated in numerous pathologies. Deficiency of NR4A2 and NR4A3 in human and murine myeloid cells reveals that both receptors function as positive regulators of enhanced MIP-3α expression. In contrast, within the same cell types and conditions, altered NR4A activity leads to suppression of LPS-induced MCP-1 gene and protein expression. An equivalent pattern of inflammatory gene regulation is replicated in TNFα-treated myeloid cells. We show that NF-κB is the critical regulator of NR4A1–3, MIP-3α, and MCP-1 during TLR4 stimulation in myeloid cells and highlight a parallel mechanism whereby NR4A activity can repress or enhance NF-κB target gene expression simultaneously. Mechanistic insight reveals that NR4A2 does not require DNA-binding capacity in order to enhance or repress NF-κB target gene expression simultaneously and establishes a role for NF-κB family member Relb as a novel NR4A target gene involved in the positive regulation of MIP-3α. Thus, our data reveal a dynamic role for NR4A receptors concurrently enhancing and repressing NF-κB activity in myeloid cells leading to altered transcription of key inflammatory mediators.

The CCR2 Inhibitor Propagermanium Attenuates Diet-Induced Insulin Resistance, Adipose Tissue Inflammation and Non-Alcoholic Steatohepatitis.

Background and aimObese patients with chronic inflammation in white adipose tissue (WAT) have an increased risk of developing non-alcoholic steatohepatitis (NASH). The C-C chemokine receptor-2 (CCR2) has a crucial role in the recruitment of immune cells to WAT and liver, thereby promoting the inflammatory component of the disease. Herein, we examined whether intervention with propagermanium, an inhibitor of CCR2, would attenuate tissue inflammation and NASH development.MethodsMale C57BL/6J mice received a high-fat diet (HFD) for 0, 6, 12 and 24 weeks to characterize the development of early disease symptoms of NASH, i.e. insulin resistance and WAT inflammation (by hyperinsulinemic-euglycemic clamp and histology, respectively) and to define the optimal time point for intervention. In a separate study, mice were pretreated with HFD followed by propagermanium treatment (0.05% w/w) after 6 weeks (early intervention) or 12 weeks (late intervention). NASH was analyzed after 24 weeks of diet feeding.ResultsInsulin resistance in WAT developed after 6 weeks of HFD, which was paralleled by modest WAT inflammation. Insulin resistance and inflammation in WAT intensified after 12 weeks of HFD, and preceded NASH development. The subsequent CCR2 intervention experiment showed that early, but not late, propagermanium treatment attenuated insulin resistance. Only the early treatment significantly decreased Mcp-1 and CD11c gene expression in WAT, indicating reduced WAT inflammation. Histopathological analysis of liver demonstrated that propagermanium treatment decreased macrovesicular steatosis and tended to reduce lobular inflammation, with more pronounced effects in the early intervention group. Propagermanium improved the ratio between pro-inflammatory (M1) and anti-inflammatory (M2) macrophages, quantified by CD11c and Arginase-1 gene expression in both intervention groups.ConclusionsOverall, early propagermanium administration was more effective to improve insulin resistance, WAT inflammation and NASH compared to late intervention. These data suggest that therapeutic interventions for NASH directed at the MCP-1/CCR2 pathway should be initiated early.

STRONTIUM RANELATE IN THE TREATMENT OF POSTMENOPAUSAL OSTEOPOROSIS: IMPACT OF VITAMIN D RECEPTOR AND MONOCYTE CHEMOATTRACTANT PROTEIN-1 GENE POLYMORPHISMS ON BONE MINERAL DENSITY: A PILOT STUDY

Objective: to investigate the impact of VDR and MCP-1 gene polymorphisms on the efficiency of 12-month therapy with strontium ranelate (SR) in women with postmenopausal osteoporosis (OP). Subjects and methods. The investigation enrolled 34 postmenopausal women (mean age 65±8 years), who was diagnosed with OP by lumbar spine and/or proximal femur examinations using a Hologic QDR 4500W X-ray bone densitometer. The gene polymorphisms of VDR (BsmI) and MCP-1 (-2518A/G) were genotyped by polymerase chain reaction, followed by restriction fragment length polymorphism analysis. Results and discussion. The carriers of the BB and Вb genotypes of the VDR gene had a statistically significantly lower increase in the bone mineral density (BMD) of the lumbar spine than those of the homozygous recessive bb genotype (p = 0.024 and p = 0.022, respectively) and the bb genotype and the BB+Bb genotype (p = 0.004), and the carriers of the GА genotype of MCP-1 gene had a statistically significantly lower increase in the BMD of the entire proximal femur than those of the homozygous AA genotype (0.2±2.5% and 4.4±4.4%, respectively; p = 0.004) at 12 months after SR treatment. Conclusion. The findings may suggest that the BB and Bb genotypes of the VDR gene and the GA genotype of the MCP-1 gene may negatively affect the efficacy of SR in patients with OP. However, additional studies on a larger sample of patients, including those with other forms of OP, are required to confirm this assumption.

Obeticholic acid protects against carbon tetrachloride-induced acute liver injury and inflammation

The farnesoid X receptor (FXR) is a ligand-activated transcription factor that plays important roles in regulating bile acid homeostasis. The aim of the present study was to investigate the effects of obeticholic acid (OCA), a novel synthetic FXR agonist, carbon tetrachloride (CCl4)-induced acute liver injury. Mice were intraperitoneally injected with CCl4 (0.15ml/kg). In CCl4+OCA group, mice were orally with OCA (5mg/kg) 48, 24 and 1h before CCl4. As expected, hepatic FXR was activated by OCA. Interestingly, OCA pretreatment alleviated CCl4-induced elevation of serum ALT and hepatic necrosis. Moreover, OCA pretreatment inhibited CCl4-induced hepatocyte apoptosis. Additional experiment showed that OCA inhibits CCl4-induced hepatic chemokine gene Mcp-1, Mip-2 and Kc. Moreover, OCA inhibits CCl4-induced hepatic pro-inflammatory gene Tnf-α and Il-1β. By contrast, OCA pretreatment elevated hepatic anti-inflammatory gene Il-4. Further analysis showed that OCA pretreatment inhibited hepatic IκB phosphorylation and blocked nuclear translocation of NF-κB p65 and p50 subunits during CCl4-induced acute liver injury. In addition, OCA pretreatment inhibited hepatic Akt, ERK and p38 phosphorylation in CCl4-induced acute liver injury. These results suggest that OCA protects against CCl4-induced acute liver injury and inflammation. Synthetic FXR agonists may be effective antidotes for hepatic inflammation during acute liver injury.

The -2518 A/G MCP-1 and -403 G/A RANTES promoter gene polymorphisms are associated with psoriasis vulgaris.

Polymorphic variants of the genes encoding monocyte chemoattractant protein-1 (MCP-1/CCL2) and regulated upon activation normal T-cell expressed and secreted (RANTES/CCL5) and their protein serum levels have not been widely explored in psoriasis. To clarify the effect of the MCP-1 (-2518 A/G) and RANTES (-403 G/A) promoter gene polymorphisms on the risk and clinical manifestation of psoriasis. We enrolled 160 unrelated patients with psoriasis vulgaris and 160 healthy, unrelated, age- and sex-matched volunteers. The promoter gene polymorphisms were analysed using amplification refractory mutation system (ARMS)-PCR and single specific primer (SSP)-PCR. Serum levels of cytokines were measured using ELISA. The presence of the MCP-1-2518 GG genotype was statistically more frequent in patients and it was associated with an increased risk of psoriasis (OR = 1.94; P = 0.04). In patients with late-onset (≥ 40 years) psoriasis, the presence of the RANTES -403 AA genotype was statistically more frequent (OR = 3.65; P < 0.01) while -403 GG was less frequent (OR = 0.44; P < 0.01). Moreover, the A allele (AA or AG) in the -403 RANTES polymorphism was associated with an increased risk of developing severe psoriasis (OR = 2.02; P = 0.03). Serum levels of both chemokines were elevated. RANTES serum concentration was significantly higher in patients with Psoriasis Area and Severity Index > 15. The results of our analysis suggest that the -2518 A/G MCP-1 and -403 G/A RANTES promoter gene polymorphisms may be risk factors for psoriasis and may influence its clinical presentation.