MCP-1 Protein Research Articles

Recombinant Treponema pallidum protein Tp0965 activates endothelial cells and increases the permeability of endothelial cell monolayer.

The recombinant Treponema pallidum protein Tp0965 (rTp0965), one of the many proteins derived from the genome of T. pallidum subsp. pallidum, shows strong immunogenicity and immunoreactivity. In this study, we investigated the effects of rTp0965 on the endothelial barrier. Treatment of human umbilical vein endothelial cells (HUVECs) with rTp0965 resulted in increased levels of ICAM-1, E-selectin, and MCP-1 mRNA and protein expression. These increases contributed to the adhesion and chemataxis of monocytes (THP-1 cells) to HUVECs preincubated with rTp0965. In addition, rTp0965 induced reorganization of F-actin and decreased expression of claudin-1 in HUVECs. Interestingly, inhibition of the RhoA/ROCK signal pathway protected against rTp0965-induced higher endothelial permeability as well as transendothelial migration of monocytes. These data indicate that Tp0965 protein may play an important role in the immunopathogenesis of syphilis.

ACE inhibitors potently reduce vascular inflammation, results of an open proof-of-concept study in the abdominal aortic aneurysm.

BackgroundIndependent of their blood pressure lowering effect, ACE inhibitors are thought to reduce vascular inflammation. The clinical relevance of this effect is unclear with the current knowledge. Abdominal aortic aneurysms (AAA) are characterized by a broad, non-specific inflammatory response, and thus provide a clinical platform to evaluate the anti-inflammatory potential of ACE inhibitors.Methods and ResultsEleven patients scheduled for open AAA repair received ramipril (5 mg/day) during 2–4 weeks preceding surgery. Aortic wall samples were collected during surgery, and compared to matched samples obtained from a biobank. An anti-inflammatory potential was evaluated in a comprehensive analysis that included immunohistochemistry, mRNA and protein analysis. A putative effect of ACE inhibitors on AAA growth was tested separately by comparing 18-month growth rate of patients on ACE inhibitors (n = 82) and those not taking ACE inhibitors (n = 204). Ramipril reduces mRNA expression of multiple pro-inflammatory cytokines such as IL-1β, IL-6, IL-8, TNF -α, Interferon-, and MCP-1, as well as aortic wall IL-8 and MCP-1 (P = 0.017 and 0.008, respectively) protein content. The is followed by clear effects on cell activation that included a shift towards anti-inflammatory macrophage (M2) subtype. Evaluation of data from the PHAST cohort did not indicate an effect of ACE inhibitors on 18-month aneurysm progression (mean difference at 18 months: −0.24 mm (95% CI: −0.90–0.45, P = NS).ConclusionsACE inhibition quenches multiple aspects of vascular inflammation in AAA. However, this does not translate into reduced aneurysm growth.Trial RegistrationNederlands Trial Register 1345.

The number of intestinal bacteria is not critical for the enhancement of antitumor activity and reduction of intestinal toxicity of irinotecan by the Chinese herbal medicine PHY906 (KD018).

BackgroundThe four-herb Chinese medicine PHY906(KD018) has been shown to both enhance the in vivo antitumor activity of irinotecan (CPT-11) against colon cancer tumor allografts and alleviate intestinal toxicity caused by CPT-11.MethodsSince intestinal bacteria can metabolize CPT-11 and PHY906, we investigated whether intestinal bacteria play a critical role in the in vivo activity of PHY906 in murine Colon-38 tumor-bearing mice. Intestinal bacteria were depleted using streptomycin/neomycin for 10 days before and during treatment with PHY906 and/or CPT-11. qPCR using 16S DNA group-specific primers was used to quantify the levels of the major intestinal bacteria.ResultsBoth PHY906 and antibiotic treatment changed the profile of intestinal bacteria species: Lactobacillus/Enterococcus, Bacteroides, Clostridium leptum, and E. rectale/C. coccoides. Antibiotic treatment did not alter the ability of PHY906 to enhance the antitumor activity of CPT-11. Antibiotic treatment alone partially reduced animal body weight loss in CPT-11-treated mice. However, PHY906 treatment was able to protect against the body weight loss in the CPT-11/antibiotic treatment group. H&E and PCNA staining of intestine showed that antibiotic treatment partially reduced the intestinal damage caused by CPT-11 but not as effectively as PHY906 treatment. Antibiotic treatment plus PHY906 conferred the most effective protection of intestine histological structure against damage by CPT-11. Both PHY906 and antibiotic treatment inhibited CPT-11-associated inflammatory processes, including infiltration of the intestine by neutrophils, MCP1 and TNF-alpha mRNA expression in the intestine, and expression of pro-inflammatory cytokines G-CSF and MCP1 proteins in the plasma. However, whereas antibiotic treatment suppressed the mRNA expression of two important intestinal progenitor/stem cell markers, Olfm4 and Lgr5, PHY906 treatment resulted in enhanced expression of these two stem cell markers.ConclusionsAlterations in the population of intestinal bacteria did not affect the abilities of PHY906 to enhance CPT-11 antitumor activity or reduce the intestinal toxicity associated with CPT-11 treatment. The major species of intestinal bacteria do not appear to play a role in PHY906’s enhancement of the therapeutic index of CPT-11 in tumor-bearing mice. Thus, patients with different intestinal bacterial profiles may still benefit from PHY906 treatment alongside CPT-11.

Doctoral and post-doctoral research - a priority of the Romanian postgraduate education system.

November 12th 2014: the opening conference for the project “Doctoral and post-doctoral research – a priority of the Romanian postgraduate education system (Doc-Postdoc)” took place in Bucharest, Romania. The project aims to support doctoral and post-doctoral research in Romania, and is cofinanced by the European Social Fund through the Sectoral Operational Programme for Human Resources Development 2007-2013; priority axis 1. “Education and training in support for growth and development of a knowledge based society“; key area of intervention: 1.5. “Doctoral and post-doctoral programs in support of research”; contract code: POSDRU/159/1.5/S/137390. The Carol Davila University of Medicine and Pharmacy in Bucharest is the project beneficiary, partnering with two other major universities in Romania: The University “Politehnica” of Bucharest, and the University of Medicine and Pharmacy Târgu Mures. The conference presented the aims and objectives of the project, but it was also packed with scientific sessions where young PhD candidates and post-doctoral researchers had the opportunity to present the studies they are conducting within this project. The topics discussed spanned through the interdisciplinary fields of biomedicine and bioengineering. Of particular interest to the readers of the Journal GERMS, Dr. Catalin Tiliscan presented data on adipocyte dysfunction in HIV-positive patients undergoing antiretroviral therapy, his study showing that almost half of patients had hyporesistinemia, and resistin was correlated with the inflammatory profile. Also, an unfavorable immune status appeared to be inversely proportional to the values of monocyte chemoattractant protein-1 MCP-1, while leptin values were not correlated with CD4 cell count or virologic status, but rather with HDL-cholesterol and waist circumference. Dr. Ramona Popescu presented an interdisciplinary study protocol for determining the incidence of intercurrent and opportunistic infections in rheumatologic patients undergoing treatment with biologic agents. Dr. Oana Sandulescu presented the preliminary results of a study aimed to determine the capacity of staphylococcal species to adhere to and internalize in eukaryotic cells. By comparing two different types of bacterial strains, collected from patients with clinically-relevant infections and from asymptomatic nasal carriage, the study showed that despite a similar rate of internalization, staphylococci from the two groups displayed statistically significant differences regarding intracellular growth after internalization. Dr. Daniela Talapan presented significant data on the emergence of carbapenemase-producing Enterobacteriaceae, with Klebsiella spp. ranking first among bacteria with resistance to carbapenems, followed by E. coli, E. cloacae, and E. aerogenes, leaving a considerably low number of active antibiotics in play, namely colistin, tigecycline and fosfomycin. Based on the preliminary data presented at the conference, the research conducted within this project is bound to generate information with a significant clinical impact, leading to a better understanding of medical issues currently understudied, with the potential to reshape clinical practice.

P1-3-5 - Angiotensin II Type 1 Receptor Signaling Promotes Head and Neck Cancer Cells Invasive Behavior by Flj10540/Mcp-1 Pathway

Head and neck squamous cell carcinoma (HNSCC) is one of the tumors of poor prognosis despite the therapeutic advances in the past few years. It has been reported that overexpression of AT1R occurs in many human carcinomas. However, the molecular mechanisms by which angiotensin II type 1 receptor (AT1R) mediate its invasive effects in HNSCC still unclear. Here, we found that AT1R was not only found in HNSCC specimens, but also significantly correlated with advanced TNM-stage and the poor 5-year survival rate. Thus indicated that AT1R was an independent factor for HNSCC development. Functionally, AT1R could promote tumor growth in nude mice. In addition, AT1R had the abilities to stimulate cell migration and invasion in oral cancer cells through increased FLJ10540 and MCP-1 expression by Transwell chambers. Conversely, depletion of AT1R expression by siRNAs suppressed FLJ10540 and MCP-1 protein expressions. Suppression MCP-1 could cause significant inhibition on cell migratory and invasive ability of AT1R-overexpressing head and neck cancer cells. Finally, immunohistochemical and Western blotting analysis of human aggressive HNSCC specimens showed a significant positively correlation among AT1R, FLJ10540 and MCP-1 expression. These findings suggest that AT1R is not only an important prognostic factor but also a new therapeutic target in the FLJ10540/MCP-1 pathway for HNSCC treatment.

Abstract 3995: Quantitative protein profiling of tumor angiogenesis and metastasis biomarkers in mouse and human models

Abstract Tumor and stromal cells secrete a variety of proteins acting as extracellular signals and creating a supportive microenvironment for tumor development, angiogenesis, and metastasis. We used the MILLIPLEX MAP cytokine/chemokine, cancer, angiogenesis, metastasis, MMP, bone metabolism, adipocyte, and cell signaling multi-pathway panels, based on the Luminex xMAP technology, to quantitatively evaluate the protein biomarker expression profiles on cultured mouse and human tumor cells and stromal cells as well as in vivo mouse and human tumor tissues. In the mouse system, biomarker profiling was performed to characterize secreted protein levels in Lewis Lung carcinoma (LLC) cells, B16.10 melanoma cells, b.End3 endothelial cells, and LLC tumors. The in vivo characterization, using primary tumors from mice bearing metastatic LLC, has identified differential expression of a panel of biomarkers, including amphiregulin (AREG), EGF, PECAM-1, KC, MCP-1, MIP-2, MMP-12, and resistin, between the high-fat (45% fat) and AIN-93G (15% fat) fed mice (p<0.05, 8 mice per group). These putative biomarkers may be involved in molecular mechanisms of the high-fat enhanced metastasis. Cell signaling pathway analysis revealed the activation of multiple pathways (JNK, p38, ERK, and STAT) in this mouse LLC model. In the human system, a heat map was generated to summarize the secreted protein signatures and the soluble receptor tyrosine kinases (RTK) profile on a series of the cultured human tumor cells (lung, brain, breast, prostate, liver, and colon) and 3 other human cell lines, HUVEC (endothelial), THP-1 (monocyte), and HFF-1 (fibroblast). Lastly, expression profiling of tumor and adjacent normal tissues from 2 patients with metastatic breast cancer was analyzed. Elevated levels of protein biomarkers, including MDC, IL-1RA, IL-8, IP-10, MCP-1, MIP-1α, OPG, OPN, SPARC, periostin, endoglin, PLGF, VEGF-A, MMP-1, MMP-7, MMP-9, and MMP-12, were detected in the breast tumors analyzed compared to their matched adjacent normal breast tissue. In summary, our results demonstrated that multiplexing using the Luminex xMAP bead-based platform for simultaneous quantitative expression profiling is a useful proteomic research tool, providing insights into biomarkers related to tumor progression and metastasis. Citation Format: Wen-Rong Lie, Jonathan Lipsey, Tim Warmke, Lin Yan, Jehangir Mistry. Quantitative protein profiling of tumor angiogenesis and metastasis biomarkers in mouse and human models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3995. doi:10.1158/1538-7445.AM2014-3995

Effects of the mTOR inhibitor rapamycin on monocyte-secreted chemokines.

BackgroundMammalian target of rapamycin (mTOR) inhibitors, such as sirolimus and its derivative, everolimus, are potent immunosuppressive and antiproliferative drugs. Inflammatory diseases are characterized by immunological dysfunction, and monocyte recruitment underlies the mechanism of cell damage. Chemokines attract inflammatory cells to sites of inflammation. Interleukin-8 (IL-8/CXCL8); the monocyte chemoattractant protein-1 (MCP-1/CCL2); the regulated on activation, normal T cell expressed, presumably secreted protein (RANTES/CCL5); the macrophage inflammatory protein (MIP)-1α (CCL3); and MIP-1β (CCL4) are involved in the pathogenesis of inflammation. However, whether mTOR inhibitors moderate the production of chemokines in monocytes remains unclear.MethodsA human monocyte cell line, THP-1, and primary monocytes obtained from human volunteers, were stimulated using lipopolysaccharide (LPS), and then treated with sirolimus. The expression of the MCP-1, RANTES, IL-8, MIP-1α, MIP-1β, and TNF-α proteins was measured using enzyme-linked immunosorbent assays, and intracellular signalling was examined using western blotting.ResultsSirolimus significantly suppressed the LPS-induced expression of MCP-1, IL-8, RANTES, MIP-1α, and MIP-1β in the THP-1 cells and human primary monocytes. The mitogen-activated protein kinase (MAPK) inhibitors that were examined suppressed the LPS-induced expression of MCP-1, IL-8, RANTES, MIP-1α, and MIP-1β. In addition, sirolimus suppressed the LPS-induced phosphorylation of p38 and p65 in the THP-1 and human primary monocytes.ConclusionSirolimus downregulates the expression of chemokines in monocytes, including MCP-1, RANTES, IL-8, MIP-1α, and MIP-1β, by inhibiting the NF-κB-p65 and MAPK-p38 signalling pathways.

Correlation between pathological and functional changes of the retina and the expression of monocyte chemoattractant protein after retinal laser injury in mice

Objective To investigate the relationship between the pathological and functional changes of the retina and the expression of monocyte chemoattractant protein (MCP)-1 after retinal laser injury in mice.Methods A total of 116 C57BL/6 mice were randomly divided into the normal group (58 mice) and the injured group (58 mice).Retinal laser injuries were induced by Argon ion laser.At 1,3,7 days after laser injury,electroretinogram (ERG) responses were recorded to detect the function of the retina.Hematoxylin and eosin (HE) staining was performed to observe pathological changes.Quantitative real-time polymerase chain reaction (PCR) was performed to detect gene expression of MCP-1.Western blot was used to measure the protein expression of MCP-1.Results HE staining showed a progressive damage of the retinal structure.The results of ERG showed that the differences of dark-adaptive a wave (t =6.998,9.594,13.778) and b wave (t=12.089,13.310,21.989) amplitudes of 1,3 and 7 day post-injury between normal group and injured group were statistically significant (P =0.000).At 1 day post-injury,the differences of light adaptive b wave amplitudes between the two groups were statistically significant (t =8.844,P=0.000).While the differences of light-adaptive a wave amplitudes were not (t=2.659,P=0.200).At 3,7 days post-injury,the differences of a (t=3.076,7.544) and b wave amplitudes (t=10.418,8.485) between the two groups were statistically significant (P=0.000).In dark-adaptive ERG,the differences of a wave amplitudes between 1 day and 3 days (t=3.773),1 day and 7 days (t=5.070) and b wave amplitudes between 1 day and 7 days (t=4.762) were statistically significant (P＜0.01),while the differences of a wave amplitudes between the 3 days and 7 days (t=1.297) and b wave amplitudes between 1dayand 3 days (t=2.236),3 day and 7 days (t=2.526) were not significant (P=0.660,0.120,0.060).In light-adaptive ERG,the differences of a wave amplitudes between 1 day and 7 days (t=2.992)and b wave amplitudes between 1 day and 3 days (t=3.570),1day and 7 days (t=4.989) were statistically significant (P＜0.05),while the differences of a wave amplitudes between 1 day and 3 days (t=0.516),the 3 days and 7 days (t=2.475) and b wave amplitudes between 3 days and 7 days (t=1.419) were not significant (P=1.000,0.710,0.070).Quantitative real-time PCR showed that the differences of MCP-1 gene expression at 1,3 and 7 day post-injury between normal group and injured group were statistically significant (t=14.329,16.861,5.743; P＜0.05).Western blot showed that the differences of MCP-1 protein expression at 1,3 and 7 day post-injury between normal group and injured group were statistically significant (t=75.068,54.145,14.653; P＜0.05).Conclusion In the first 7 days after mice retinal laser injury,there are progressive pathological and functional damage of the retina,which might be correlated with MCP-1 expression. Key words: Retina diseases/etiology; Electroretinography; Chemokine CCL2; Animal experimentation

Effects of β-aescine sodium plus oxiracetam on plasma homocysteine and MCP-1 protein in patients with acute cerebral infarction

Objective To study the effects of β-aescine sodium plus oxiracetam on plasma homocysteine (Hcy) and monocyte chemotactic factor(MCP-1) protein in patients with acute cerebral infarction.Methods 117 patients with acute cerebral infarction were randomly divided into the two groups,the observation group (n =60 cases) and the control group (n =57 cases).The patients in the control group were treated with the conventional treatment,while the patients in the observation group were treated with joint treatment ofβ-aescine sodium plus oxiracetam.They were treated for 14 days.Plasma Hcy and MCP-1 were detected.Results The total efficicies were 95.0％ in the observation group and 84.5％ in the control group.There was a significant difference between two groups (x2 =5.801,P ＜0.05).Plasma Hcy and MCP-1 were decreased after treatment (t =9.551,4.296,2.001,21.070,P ＜ 0.05,P ＜0.01).Plasma Hcy and MCP-1 in the observation group were lower than these in the control group (t =4.997,25.276,all P ＜ 0.05).Conclusion β-aescine sodium plus oxiracetam can decrease plasma Hcy and MCP-1 in patients with acute cerebral infarction. Key words: Cerebral infarction; Oxiracetam; β-aescine sodium; Homocysteine; Monocyte chemotactic factor

Constitutive and LPS-induced expression of MCP-1 and IL-8 by human uveal melanocytes in vitro and relevant signal pathways.

Melanocytes are one of the major cellular components in the uvea. Interleukin-8/CXCL8 and monocyte chemoattractant protein-1 (MCP-1/CCL2) are the two most important proinflammatory chemokines. We studied the constitutive and lipopolysaccharide (LPS)-induced expression of IL-8 and MCP-1 in cultured human uveal melanocytes (UM) and explored the relevant signal pathways. Conditioned media and cells were collected from UM cultured in medium with and without stimulation of LPS. Interleukin-8 and MCP-1 proteins and mRNAs were measured using an ELISA kit and RT-PCR, respectively. Nuclear factor (NF)-κB in nuclear extracts and phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases1/2 (ERK1/2), and c-Jun N-terminal kinase1/2 (JNK1/2) in cells cultured with and without LPS were measured by ELISA kits. Inhibitors of p38 (SB203580), ERK1/2 (UO1026), JNK1/2 (SP600125), and NF-κB (BAY11-7082) were added to the cultures to evaluate their effects. Low levels of IL-8 and MCP-1 proteins were detected in the conditioned media in UM cultured without serum. Lipopolysaccharide (0.01-1 μg/mL) increased IL-8 and MCP-1 mRNAs and proteins levels in a dose- and time-dependent manner, accompanied by a significant increase of phosphorylated JNK1/2 in cell lysates and NF-κB in nuclear extracts. Nuclear factor-κB and JNK1/2 inhibitors significantly blocked LPS-induced expression of IL-8 and MCP-1. This is the first report on the expression and secretion of chemokines by UM. The data suggest that UM may play a role in the pathogenesis of ocular inflammatory diseases.

Targeting complement anaphylatoxin C5a receptor in hyperoxic lung injury in mice.

Receptor binding of complement anaphylatoxin C5a results in proinflammatory activation of numerous diseases, but the role of receptor-mediated action during hyperoxic lung injury has, to the best of our knowledge, not yet been investigated. The contribution of the C5a receptor (C5aR) to hyperoxic lung injury in mice was investigated in this study. The effect of C5aR on hyperoxic lung injury in Balb/c mice was examined employing a C5aR antagonist (C5aRA). The mice were ventilated with 100% oxygen for 36 h with or without the administration of C5aRA. C5aR expression levels in non-treated or 100% oxygen-treated mice were assessed by reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. The body weight and the relative lung weight of the mice, and the morphological changes in the lung were then determined. The total cell counts and the number of macrophages, neutrophils and lymphocytes in bronchoalveolar lavage fluid (BALF) were determined using cytocentrifuge slides and a hemocytometer. The levels of interleukin-6 (IL-6), monocyte chemotactic protein (MCP-1) and tumor necrosis factor-α (TNF-α) in BALF and the myeloperoxidase (MPO) activity in the lung tissue were measured by enzyme-linked immunosorbent assay. The relative levels of CD68 and F4/80 messenger ribonucleic acid (mRNA) expression in the lung tissue were detected by RT-PCR. The TNF-α, IL-6 and MCP-1 protein expression levels in the lung tissue were assessed by western blot analysis. The results revealed hyperoxia-induced morphological changes, lung injury and increased expression levels of C5aR in lung tissue. The hyperoxia-induced increases in the total cell count and the number of macrophages, neutrophils and lymphocytes in the BALF were all significantly reduced in the mice receiving C5aRA. Treatment with C5aRA also attenuated the morphological changes and reduced MPO activity, and CD68 and F4/80 mRNA expression levels in the lung tissue, as well as the levels of IL-6, MCP-1 and TNF-α in BALF and lung tissue. In conclusion, C5a-C5aR action accelerated hyperoxia-induced lung injury, but this hyperoxic lung injury was attenuated by treatment with C5aRA.

Maternal intravenous treatment with either azithromycin or solithromycin clears Ureaplasma parvum from the amniotic fluid in an ovine model of intrauterine infection.

Intrauterine infection with Ureaplasma spp. is strongly associated with preterm birth and adverse neonatal outcomes. We assessed whether combined intraamniotic (IA) and maternal intravenous (IV) treatment with one of two candidate antibiotics, azithromycin (AZ) or solithromycin (SOLI), would eradicate intrauterine Ureaplasma parvum infection in a sheep model of pregnancy. Sheep with singleton pregnancies received an IA injection of U. parvum serovar 3 at 85 days of gestational age (GA). At 120 days of GA, animals (n=5 to 8/group) received one of the following treatments: (i) maternal IV SOLI with a single IA injection of vehicle (IV SOLI only); (ii) maternal IV SOLI with a single IA injection of SOLI (IV+IA SOLI); (iii) maternal IV AZ and a single IA injection of vehicle (IV AZ only); (iv) maternal IV AZ and a single IA injection of AZ (IV+IA AZ); or (v) maternal IV and single IA injection of vehicle (control). Lambs were surgically delivered at 125 days of GA. Treatment efficacies were assessed by U. parvum culture, quantitative PCR, enzyme-linked immunosorbent assay, and histopathology. Amniotic fluid (AF) from all control animals contained culturable U. parvum. AF, lung, and chorioamnion from all AZ- or SOLI-treated animals (IV only or IV plus IA) were negative for culturable U. parvum. Relative to the results for the control, the levels of expression of interleukin 1β (IL-1β), IL-6, IL-8, and monocyte chemoattractant protein 2 (MCP-2) in fetal skin were significantly decreased in the IV SOLI-only group, the MCP-1 protein concentration in the amniotic fluid was significantly increased in the IV+IA SOLI group, and there was no significant difference in the histological inflammation scoring of lung or chorioamnion among the five groups. In the present study, treatment with either AZ or SOLI (IV only or IV+IA) effectively eradicated macrolide-sensitive U. parvum from the AF. There was no discernible difference in antibiotic therapy efficacy between IV-only and IV+IA treatment regimens relative to the results for the control.

Abstract 374: Inhibitor of Differentiation 3 Promotes High Fat--Induced Expansion of Committed Inflammatory Adipocyte Progenitor Cells

Background: Global deletion of helix-loop-helix (HLH) transcription factor Inhibitor of Differentiation 3 (Id3) attenuates high fat diet (HFD) induced obesity and adipose tissue inflammation. Specifically, Id3 -/- mice have less epididymal adipose tissue (eAT), fewer proinflammatory macrophages in eAT after HFD, and attenuated HFD-induced levels of MCP-1 mRNA and protein in eAT. It is currently unclear how Id3 is regulating MCP-1 or macrophage accumulation in eAT. Methods & Results: Id3 -/- mice and Id3 +/+ littermates were fed 60% HFD (n=7, 5) or standard chow (n=6, 7) for 1 week. Intracellular levels of MCP-1 in stromavascular fraction (SVF) cells from eAT were analyzed via flow cytometry. HFD increased the number of MCP-1 hi cells in Id3 +/+ mice compared to chow controls (8.66 vs 2.36 x10 4 , p<0.05), but there was no increase in Id3 -/- mice (3.28 vs 1.53 x10 4 , n.s.). Phenotyping of the SVF by flow cytometry revealed that MCP-1 hi cells were mostly adipocyte progenitor cells (AdPCs) (78.8±6.8%), recently characterized as CD31 - CD45 - Ter119 - CD34 + CD29 + Sca-1 + . Interestingly, HFD induced an Id3-dependent increase in AdPCs. It was recently shown that CD24 - AdPCs are more committed to the adipocyte lineage than CD24 + AdPCs. CD24 + AdPCs lacked MCP-1 expression, but committed CD24 - AdPCs expressed MCP-1. To determine proliferation of AdPCs, BrdU was i.p. injected during the 1 week HFD ( Id3 +/+ n=11, Id3 -/- n=7). Id3 +/+ mice had a HFD-induced increase in proliferating AdPCs (11.71% vs 5.71%, p<0.01), while, Id3 -/- mice lacked an increase (7.96% vs 6.69%, n.s.). Finally, OP-9 and 3T3-L1 preadipocyte cell lines were transfected with a p21 Cip1 luciferase promoter construct. Co-transfection with Id3 reduced p21 Cip1 promoter activation by 2-fold (p<0.01). Conclusion: Committed AdPCs are the predominant source of MCP-1 in the early stages of high fat feeding, and diet-induced expansion of these AdPCs is dependent on Id3. Id3 inhibition of p21 Cip1 promoter activation in AdPCs may be one potential mechanism mediating this effect. This novel evidence of the inflammatory properties of AdPCs and the role that Id3 plays in their expansion during obesity may lead to new therapeutic targets for the treatment of diet-induced adipose inflammation and its metabolic consequences.

Abstract 662: Maternal Soy Protein Diet Ameliorates Atherosclerotic Lesions in ApoE-Deficient Mice Offspring by Promoting Anti-inflammatory Responses Through Epigenetic Changes

Maternal hypercholesterolemia has been implicated with an earlier onset of atherosclerotic lesions in neonatal offspring. In this study, we investigated whether gestational exposure to soy protein isolate (SPI) diet (maternal diet) primes the vessel wall to attenuate development of atherosclerosis in adult F1 offspring. Pregnant apolipoprotein E knockout (apoE-/-) female mice were fed SPI diet until postnatal day 21 (PND21) of the offspring (SPI-offspring). At PND21, SPI-offspring was switched to casein (CAS) diet until PND140. Mice fed CAS throughout the lifetime (gestation to adult) were used as control (CAS-offspring). Atherosclerotic lesions in aortic sinus were reduced in SPI-offspring compared with CAS-offspring. Total cholesterol levels in CAS- or SPI-offspring, and CAS- or SPI-fed dams were not different suggesting that alternative mechanism(s) contributes to the athero-protective effect of gestational SPI diet. Aortic VCAM-1, MCP-1 and TNF-α mRNA and protein expression were elevated in CAS offspring, while expression of these molecules were 50% reduced in SPI-offspring. Interestingly, compared to CAS-offspring IFN-γ expression (Th1) was reduced, while expression of IL-10 (Th2/Treg), and IL-13 (Th2) expression was increased in SPI-offspring, suggesting that Th2/Treg bias could have contributed to the athero-protective effect. DNA methylation analyses revealed gata3 and il13 promoter regions were hypomethylated in SPI-offspring. We conclude that in utero soy protein exposure inhibits susceptibility to atherosclerotic lesion formation by promoting anti-inflammatory responses through epigenetic regulation.

Expression and cellular distribution of TLR4, MyD88, and NF-κB in diabetic renal tubulointerstitial fibrosis, in vitro and in vivo

AimInflammation and extracellular matrix hyperplasia are crucial in the pathogenesis of tubulointerstitial fibrosis (TIF) involved in diabetic nephropathy (DN). Macrophage accumulation plays a major role, but whether immune factors contribute to DN pathogenesis is not well understood. This study aimed to investigate TLR4-MyD88-NF-κB-dependent pathway's involvement in TIF pathogenesis. MethodsSTZ-induced diabetic rats and rat renal tubular epithelial NRK-52E cells cultured under high glucose conditions were used as in vivo and in vitro models, respectively. Real-time RT-PCR, western blot, immunohistochemistry and immunofluorescence were performed to examine the mRNA and protein levels of TLR4, MyD88, NF-κB, MCP-1, and α-SMA. ResultsCompared with 5.5mmol/L glucose, treatment of NRK-52E cells with 25 and 50mmol/L d-glucose resulted in significantly increased TLR4 and MyD88 mRNA and protein levels (P<0.05). TLR4 and MyD88 were detected in the cytoplasm of most NRK-52E cells cultured under high glucose. Pronounced damage in the renal tubulointerstitium was observed in diabetic rats (scores: 3.82±0.65 vs. 0.38±0.08, P<0.01). Compared with the normal controls, a sharp upregulation of TLR4, MyD88, NF-κB p65, MCP-1, and α-SMA mRNA and protein levels was observed in diabetic rat kidneys (P<0.05). In diabetic animals, TLR4 and MyD88 were strongly expressed in the cytoplasm, while NF-κB p65 was widely expressed in cytoplasm and nuclei of renal tubular epithelial cells. ConclusionThe inflammatory reaction and epithelial-mesenchymal transformation observed in renal tubulointerstitium may be the result of overactivation of the TLR4-MyD88-NF-κB-dependent innate immunity under high glucose, and may be involved in DN occurrence and progression.

NanoSOD reduces adipose tissue inflammation in obesity (1107.10)

Obesity‐linked hypertension is resistant to antihypertensive drugs and, adipose tissue (AT) inflammation is considered to mediate the pathological consequences of obesity. The objective of this study is to determine the effectiveness of nanoformulated copper/zinc superoxide dismutase (nanoSOD) in reducing AT inflammation in obesity. Wild type mice were fed a high fat (HF) diet (45% fat) for 8 or 13 wk followed by 2 wk treatment with native or nanoSOD. The drug uptake studies showed that nanoSOD was accumulated in the visceral AT to a greater extent than native SOD in obese mice. The mice fed a HF diet for 8 wk followed by 2 wk treatment with nanoSOD showed an increase in SOD content in stromal vascular cells (SVCs) collected from AT and in the plasma. These mice also showed a reduction in nitrotyrosine level, a marker of superoxide induced oxidative stress, in SVCs. The AT inflammatory markers showed a trend towards a decrease. The serum toxicity markers did not alter in nanoSOD compared to other groups suggesting that the nanoSOD is safe to use in vivo. After 13 wk on a HF diet followed by 2 wk treatment, the SVCs derived from AT of nanoSOD treated mice showed a decrease in the mRNA levels of MCP‐1 and MIP‐1α. Also, the MCP‐1 mRNA and protein levels in total AT was reduced to a greater extent in nanoSOD compared to native SOD treated mice. Our data suggest that nanoSOD may be a potential therapeutic agent to ameliorate obesity‐linked cardiovascular risk factors, in particular, systemic hypertention.Grant Funding Source: National Institutes of Health (NIH‐NIGMS)

Cartilage injury regulates nerve growth factor (NGF) in vitro and in vivo and drives painful behaviour in murine OA

Cartilage injury regulates nerve growth factor (NGF) in vitro and in vivo and drives painful behaviour in murine OA

Immediate effects of varus bracing on knee mechanics in people with predominant lateral knee osteoarthritis and valgus malalignment, 12 years after anterior cruciate ligament reconstruction

Immediate effects of varus bracing on knee mechanics in people with predominant lateral knee osteoarthritis and valgus malalignment, 12 years after anterior cruciate ligament reconstruction

Bone invasion in oral squamous cell carcinoma (OSCC)

Background: Bone invasion in OSCC has poor outcomes. We hypothesise stimulation of osteoclasts directly by cancer cells; indirectly via osteoblasts. Methods and Results: Cross-over co-culture of OSCC and bone cell-lines shows: matrix-metalloproteinases (MMPs) from OSCC affect osteoblast differentiation and osteoclastogenesis; conditioned medium (CM) from osteoblasts increases MMPs in OSCC cells. Western blotting showed increased Twistl protein (not Runx2). RANKL/OPG ratio, zymogen and protein expression of MMP-9 were increased in bone cells cultured with CM from OSCC cell lines; rtPCR shows mRNA consistent with protein. All these molecules were expressed in invading malignant keratinocytes; all but OPG in osteoclasts. OSCC cell-lines were induced to epithelial-mesenchymal-transition (EMT) by TGF-β. Whilst this did not affect proliferation, Vimentin was induced and EMT markers Twist1 and N-cadherin up-regulated; Snaill and E-cadherin down-regulated. Of factors associated with invasion, MMP-2,9 were increased/unchanged in OSCC cell-lines. For osteo-clast-related molecules, membrane-type-1-MMP and RANKL were up-regulated; OPG down-regulated in all cells. CM from OSCC cells pre-treated with TGF-β prolonged survival of mature osteoclasts. For future biotherapies, we asked whether inhibition of monocyte-chemotactic-protein (MCP)-l, a chemokine which recruits/activates osteoclasts, might inhibit bone invasion. MCP-1 protein was observed in tumour cells and osteoclasts from 10 patients. Real-time-PCR demonstrated MCP-1 mRNA highly expressed by OSCC cell-lines. A plasmid with inhibitor of MCP-1 (7ND vector) was transfected into SCC25 cells: CM of SCC25-7ND efficiently inhibited formation of human osteoclasts from a CD14+ monocyte subpopulation. Conclusion: Extensive crosstalk occurs between cancer and bone cells, suggesting pathways for inhibition of bone invasion.

Mizoribine selectively attenuates monocyte chemoattractant protein-1 production in cultured human glomerular mesangial cell: A possible benefit of its use in the treatment of lupus nephritis

Mizoribine (MZR) is a selective inhibitor of the inosine monophosphate dehydrogenase - a key enzyme in the de novo pathway of guanine nucleotides - that was developed in Japan. Besides its immunosuppressive effects, MZR has recently been reported to suppress the progression of histologic chronicity via suppression of macrophage infiltration of the interstitium in selected patients with lupus nephritis. We examine the direct effect of MZR in human mesangial cells on the expression of functional molecules including monocyte chemoattractants in cultured human mesangial cells (MCs) treated with polyinosinic-polycytidylic acid (poly IC), a synthetic analogue of viral dsRNA, that makes 'pseudoviral' infection, and analyzed the expression of target molecules by reverse transcriptase-polymerase chain reaction and Western blotting. Thereafter, the effect of MZR on the expressions was examined. Pretreatment of cells with MZR partially, but significantly, attenuates the expression of monocyte chemoattractant protein (MCP)-1 mRNA and protein, whereas the poly IC-induced expressions for the other functional molecules, such as CCL5, fractalkine and IL-8 were not influenced by MZR treatment. On the other hand, pretreatment of cells with tacrolimus did not suppress the expression of MCP-1 mRNA. Mizoribine itself selectively attenuated the expression of MCP-1 both mRNA and protein levels in MCs treated with poly IC; that is, a possible model of 'pseudoviral' infection, which may be involved in the pathogenesis of lupus nephritis.