MCP-1 Protein Expression Research Articles

Hepatoprotective effect of peony total glucosides and the underlying mechanisms in diabetic rats

Context: Total glucosides of peony (TGP), compounds extracted from the dried roots of Paeonia lactiflora Pall, have been reported to have anti-inflammatory and antioxidative activities. However, the protective effect of TGP on liver injury and the underlying mechanisms remains unknown in diabetic rats. Objectives: Current study investigates prevention of liver injury by TGP in diabetic rats and its mechanism was related to the inhibition of endoplasmic reticulum stress (ERS). Materials and methods: Fifty adult male rats were randomly divided into: Normal group, diabetic group, TGP (50, 100 and 200 mg/kg/day) treatment groups (n = 10 per group). At the end of the 8th week, the liver was removed for biochemical and histological examinations. Results: Compared with the diabetic group, administration of TGP at doses of 50, 100 and 200 mg/kg significantly prevented the increase of hepatic fibrosis score (ED50 139.4 mg/kg). Compared with diabetic group, TGP at doses of 50, 100 and 200 mg/kg showed an inhibition on the increased macrophage infiltration. MCP-1 and TNF-α mRNA and protein expression were significantly increased in diabetic group compared with normal group; TGP administration caused significant reduction of high levels of MCP-1 and TNF-α mRNA as well as protein levels. Also, TGP at all doses showed an inhibition on the increased GRP78 levels, p-Perk levels and p-Eif2α levels in liver from diabetic group. Discussion and conclusions: Our results indicate that TGP has potential as a treatment for diabetic liver injury attenuating liver lipid accumulation and inflammation as well as ERS induced by diabetic condition.

Antagonistic effects of endostatin-vascular endothelial growth inhibitor chimeric recombinant adenovirus on homocysteine-induced vascular endothelial cells injury in vitro and in vivo.

Background:The study is inclined to investigate the antagonistic effects of endostatin-vascular endothelial growth inhibitor chimeric recombinant adenovirus (Ad-hENDO-VEGI) on homocysteine (Hcy)-induced vascular endothelial cells (VECs) injury in vitro and in vivo.Methods:Human VECs cell line ECV304 was selected and infected with Ad-hENDO-VEGI. The LDH leakage, SOD activity, and MDA levels were measured by the automatic biochemical analyzer. Cell survival rate was counted by Trypanblau dying. The TNF-α and MCP-1 protein expressions were detected by ELISA assay. The protein expressions of fusion protein of Ad-hENDO-VEGI, nuclear factor kappa B p65 (NF-kappa B p65), and NF-kappa B inhibitor alpha (I-kappa B-α) were detected by Western blotting. A rat model of hyper-homocysteinemia was constructed. Thirty-six Wistar rats were randomly divided into 3 groups: the control group, the model group, and the Ad-hENDO-VEGI group. Serum Hcy levels in rats were measured with enzymatic cycling method. Endothelial vasodilation function was evaluated with the treatment of sodium nitroprusside and acetylcholine.Results:After Ad-hENDO-VEGI infection, high expressions (41 kD) of fusion proteins in ECV304 cells were observed. The injury severity of Hcy on ECV304 cells had a dose-dependent manner, and the injury reached a steady stage at 1.0 mmol/L. Thus, 1.0 mmol/L Hcy was selected for further experiments. With an increase of Ad-hENDO-VEGI in ECV304 cells after Hcy treatment, LDH leakage, MDA, TNF-α, MCP-1, and nuclear NF-kappa B p65 protein expression were gradually decreased, and cell survival rate, SOD activity, and I-kappa B-α protein expression were gradually increased. Compared with the control group, the model group had a higher Hcy level and attenuated vasodilator response. The Ad-hENDO-VEGI group exhibited a lower Hcy level and enhanced vasodilator response than the model group.Conclusion:These results indicated that Ad-hENDO-VEGI could down-regulate NF-kappa B p65 expression and suppress inflammatory response, thereby alleviating Hcy-induced VECs injury.

Clarithromycin attenuates the expression of monocyte chemoattractant protein-1 by activating toll-like receptor 4 in human mesangial cells.

Signaling pathways induced by the activation of renal toll-like receptor 4 (TLR4) play a pivotal role in chronic kidney disease (CKD). Some recent studies suggested that clarithromycin (CAM), a 14-membered ring macrolide, exerts renoprotective effects by suppressing proinflammatory chemokines. However, its beneficial effects on signaling pathways through renal TLR4 activation are unknown. Cultured human mesangial cells (MCs) were treated with lipopolysaccharide (LPS). Expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) and interleukin-8 (IL-8/CXCL8) was analyzed by quantitative RT-PCR and enzyme-linked immunosorbent assay. Signaling pathways affected by CAM were determined by examining the activation of nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase (MAPK) by performing western blotting. CAM inhibited both the mRNA and protein expression of MCP-1 without cell injury but did not affect those expressions of IL-8 in LPS-stimulated MCs. Interestingly, CAM decreased p38 MAPK activation by inhibiting phosphorylation but did not affect NF-κB activation. Our results indicated that CAM exerted renoprotective effects by suppression of p38 MAPK activity and by decreasing the expression of MCP-1 in LPS-stimulated MCs. Given the implication of TLR4 signaling in CKD, CAM may be a potential treatment of choice for CKD.

Effect of the oral iron chelator deferiprone in diabetic nephropathy rats.

The aim of the present study was to investigate the effects of the iron chelator deferiprone in diabetic nephropathy (DN) rats and the mechanisms involved. Thirty-two male Wistar rats (180-220 g, 6 weeks old) were randomly divided into a control group, a DN group and two DN groups treated with either 50 or 100 mg/kg per day deferiprone. The DN group was established by feeding of a high-carbohydrate-fat diet and injection of 35 mg/kg streptozotocin into the vena caudalis. The duration of deferiprone treatment was 20 weeks. Histopathological changes were detected by hematoxylin-eosin and Masson staining, as well as transmission electron microscopy. Levels of nuclear factor (NF)-κB, monocyte chemotactic protein (MCP)-1, matrix metalloproteinase (MMP)-9, tissue-specific inhibitor of metalloproteinase (TIMP)-1, cyclo-oxygenase (COX)-2, and nitrotyrosine were determined in kidney tissues using reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and immunohistochemistry. Histopathological observations showed that deferiprone treatment alleviated inflammation infiltrates and collagenous fibrosis in DN rats. Results from RT-PCR and western blotting indicated that deferiprone inhibited the expression of NF-κB, MCP-1, COX-2, and nitrotyrosine, which were overexpressed in DN rats. Immunohistochemistry showed that the mechanism of deferiprone action may involve regulation of MMP-9 and TIMP-1. Decreased MMP-9 expression and increased TIMP-1 expression in DN rats were significantly promoted and inhibited by deferiprone, respectively. Iron chelation by oral deferiprone has a renoprotective effect in DN rats by relieving oxidative stress, inflammation, and fibrosis, which is related to the cytokines NF-κB, MCP-1, MMP-9, TIMP-1, COX-2, and nitrotyrosine.

Human β-defensin-3 alleviates the progression of atherosclerosis accelerated by Porphyromonas gingivalis lipopolysaccharide

Background and aimPorphyromonas gingivalis (P.gingivalis) lipopolysaccharide (LPS) is reported to be associated with the progression of atherosclerosis (AS). In this study, we explored the potential of human β-defensin-3 (hBD3), an antimicrobial peptide with immunomodulatory properties, to alleviate AS progression accelerated by P.gingivalis LPS and the mechanism underlying this effect. Materials and methodsApolipoprotein E-deficient mice were injected intraperitoneally with hBD3, P.gingivalis LPS, or hBD3+P.gingivalis LPS. The aorta was assessed immunohistologically and mRNA levels of inflammatory cytokines were determined by quantitative PCR. Macrophages and vascular endothelial cells were stimulated in vitro to investigate the hBD3 target cells. Inflammatory cytokines in serum and cell culture supernatants were detected using cytometric bead arrays. Signaling pathways were investigated by Western blotting. ResultsIn P.gingivalis LPS-treated mice, hBD3 significantly reduced serum IL-6 and TNF-α levels and aortic expression of ICAM-1, IL-6, and MCP-1 (mRNA and protein). The area and severity of atherosclerotic lesions were also diminished, with less advanced plaque formation, more continuous and distinct elastic lamina, and more normal smooth muscle cells arranged along the tunica media layer. In vitro, hBD3 decreased TNF-α, IL-1β, IL-6 secretion and downregulated TNF-α, IL-1β, IL-6, IL-8, VCAM-1, and IL-10 mRNA levels in macrophages. hBD3 did not influence TNF-α, IL-6, and IL-8 levels in HUVECs culture supernatants. Furthermore, hBD3 suppressed P.gingivalis LPS-induced activation of the NF-κB, p38 and JNK pathways. ConclusionhBD3 alleviates AS progression accelerated by P.gingivalis LPS in apolipoprotein E-deficient mice by downregulating the cytokine expression in macrophages via the MAPK and NF-κB signaling pathways.

Curcumin improves LPS-induced preeclampsia-like phenotype in rat by inhibiting the TLR4 signaling pathway

IntroductionAbnormal inflammation mediated by Toll-like receptor 4 (TLR4) signaling pathway contributes to preeclampsia (PE). Because curcumin can inhibit TLR4 signaling pathway, we investigated its effects on a PE rat model. MethodsTwenty-one pregnant rats were randomly divided into three groups: 1) seven rats were injected 0.5 μg/kg lipopolysaccharide (LPS) on gestational day (GD) 5 to create a PE model (LPS-treated group), 2) seven rats were injected with a similar dosage of LPS and further treated with curcumin (0.36 mg/kg) (LPS-curcumin-treated group), 3) seven rats received saline (control group). Blood pressure and urinary protein level were observed. Immunostaining and periodic acid-Schiff staining of placenta were conducted. TLR4 and downstream Nuclear Factor-κB (NF-κB) expressions of placenta were analyzed by Western blot and immunohistochemistry. IL-6 and MCP-1 in rat serum and placenta were determined by ELISA and qRT-PCR. ResultsCompared to LPS-treated group, LPS-curcumin-treated group had decreased blood pressure and urinary protein level, similar to control group. Furthermore, deficient trophoblast invasion and spiral artery remodeling induced by LPS were improved by curcumin. Increased TLR4, NF-κB and IL-6, MCP-1 protein expressions in LPS-treated group were significantly decreased after curcumin administration. DiscussionCurcumin improves the PE-like phenotype in rat model by reducing abnormal inflammation related to TLR4 signaling pathway.

Emodin suppresses LPS-induced inflammation in RAW264.7 cells through a PPARγ-dependent pathway

Inflammation is a defense and protective response to multiple harmful stimuli. Over and uncontrolled inflammation can lead to local tissues or even systemic damages and injuries. Actually, uncontrolled and self-amplified inflammation is the fundament of the pathogenesis of a variety of inflammatory diseases, including sepsis shock, acute lung injury and acute respiratory distress syndrome (ALI/ARDS). Our recent study showed that emodin, the main active component of Radix rhizoma Rhei, could significantly ameliorate LPS-induced ALI/ARDS in mice. However, its underlying signal pathway was not still very clear. Then, the aim of current study was to explore whether emodin could attenuate LPS-induced inflammation in RAW264.7 cells, and its involved potential mechanism. The mRNA and protein expression of ICAM-1, MCP-1 and PPARγ were measured by qRCR and western blotting, the production of TNF-α was evaluated by ELISA. Then, the phosphorylation of NF-κB p65 was also detected by western blotting. And NF-κB p65 DNA binding activity was analyzed by ELISA as well. Meanwhile, siRNA-PPARγ transfection was performed to knockdown PPARγ expression in cells. Our data revealed that LPS-induced the up-regulation of ICAM-1, MCP-1 and TNF-α, LPS-induced the down-regulation of PPARγ, and LPS-enhanced NF-κB p65 activation and DNA binding activity were substantially suppressed by emdoin in RAW264.7 cells. Furthermore, our data also figured out that these effects of emdoin were largely abrogated by siRNA-PPARγ transfection. Taken together, our results indicated that LPS-induced inflammation were potently compromised by emodin very likely through the PPARγ-dependent inactivation of NF-κB in RAW264.7 cells.

PGC-1β suppresses saturated fatty acid-induced macrophage inflammation by inhibiting TAK1 activation.

Inflammation of infiltrated macrophages in adipose tissue is a key contributor to the initiation of adipose insulin resistance. These macrophages are exposed to high local concentrations of free fatty acids (FFAs) and can be proinflammatory activated by saturated fatty acids (SFAs). However, the regulatory mechanisms on SFA-induced macrophage inflammation are still elusive. Peroxisome proliferator-activated receptor γ coactivator-1β (PGC-1β) is a member of the PGC-1 family of transcriptional coactivators and has been reported to play a key role in SFAs metabolism and in the regulation of inflammatory signaling. However, it remains unclear whether PGC-1β is involved in SFA-induced macrophage inflammation. In this study, we found that PGC-1β expression was significantly decreased in response to palmitic acid (PA) in macrophages in a dose dependent manner. PGC-1β inhibited PA induced TNFα, MCP-1, and IL-1β mRNA and protein expressions. Furthermore, PGC-1β significantly antagonized PA induced macrophage nuclear factor-κB (NF-κB) p65 and JUN N-terminal kinase activation. Mechanistically, we revealed that TGF-β-activated kinase 1 (TAK1) and its adaptor protein TAK1 binding protein 1 (TAB1) played a dominant role in the regulatory effects of PGC-1β. We confirmed that PGC-1β inhibited downstream inflammatory signals via binding with TAB1 and thus preventing TAB1/TAK1 binding and TAK1 activation. Finally, we showed that PGC-1β overexpression in PA treated macrophages improved adipocytes PI3K-Akt insulin signaling in a paracrine fashion. Collectively, our results uncovered a novel mechanism on how macrophage inflammation induced by SFAs was regulated and suggest a potential target in the treatment of obesity induced insulin resistance.

Effects of JAZF1 overexpression on proinflammatory cytokines in hepatocytes induced by palmitic acid

To investigate the effects of JAZF1 overexpression on the pro-inflammatory cytokines in hepatic steatosis. The model of hepatic steatosis was established by incubating hepatocytes with palmitic acid (PA) at 0, 0.125, 0.25, 0.5 and 1 mM dose and for 0, 6, 12, 24 and 48 hours, after which recombinant adenovirus expressing JAZF1 (Ad-JAZF1) was introduced to up-regulate expression. Triglyceride level was measured by GOD. Cell viability was detected by CCK-8. The mRNA and protein expression of TNF-alpha, MCP-1, IL-8 and JAZF1 was examined by RT-PCR, ELISA, and western blotting. The PA-treated hepatocytes showed dose-dependent significant increases in TNF-alpha, MCP-1 and IL-8 mRNA expression for doses up to 0.25 mM; there were no significant increases for the highest doses of 0.5 and 1 mM. The 0.25 mM PA-treated hepatocytes showed time-dependent significant increases in TNF-alpha, MCP-1 and IL-8 mRNA expressions (FTNF-alpha = 26.51, FMCP-1 = 57.20, FIL-8 = 353.85, P less than 0.01), with the maximum level reached at 12 h and followed by a gradual decrease with longer treatment times. JAZF1 mRNA and protein expression was markedly increased in hepatocytes infected with Ad-JAZF1 (P less than 0.01). However, the AP-treated hepatocytes with JAZF1 overexpression showed down-regulation of TNF-alpha, MCP-1 and IL-8 mRNA expression (decreased by 89.69%, 77.68%, and 83.21%, respectively) and secretion (37%, 37% and 41%, respectively, P less than 0.01). Stimulation of hepatocytes by the PA fatty acid in vitro promotes mRNA expression of TNF-alpha, MCP-1 and IL-8, but overexpression of JAZF1 inhibits the PA-induced expression and secretion of these factors.

IL-1β-induced MCP-1 expression and secretion of human dental pulp cells is related to TAK1, MEK/ERK, and PI3K/Akt signaling pathways

ObjectiveInterleukin-1β (IL-1β) is an inflammatory molecule of the dental pulp. IL-1β stimulates cyclooxygenase-2 (COX-2) and prostaglandins production of pulp cells and affects the pulpal inflammation and repair. However, the effects of IL-1β on Monocyte Chemotactic Factor-1 (MCP-1) of dental pulp cells and its relation to transforming growth factor β-activated kinase-1 (TAK1), PI3K/Akt, and MEK/ERK signaling and COX activation are not fully clear. DesignHuman dental pulp cells were exposed to IL-1β with/without pretreatment and co-incubation by aspirin (a COX inhibitor), 5z-7-oxozeaenol (a TAK1 inhibitor), LY294002 (a PI3K/Akt inhibitor) or U0126 (a MEK/ERK inhibitor). Viable cell number was evaluated by MTT assay. MCP-1 mRNA expression was tested by reverse transcriptase-polymerase chain reaction (RT-PCR). MCP-1 and COX-2 protein expression was studied by western blot. MCP-1 in the culture medium was measure by ELISA. ResultsIL-1β showed little cytotoxicity to pulp cells. It stimulated MCP-1 mRNA and protein expression and MCP-1 secretion. Aspirin, U0126, LY294002 and 5z-7-oxozeaenol attenuated the IL-1β-induced MCP-1 expression. In addition, 5z-7-oxozeaenol, LY294002, U0126 and aspirin prevented the IL-1β-induced MCP-1 secretion of pulp cells. ConclusionThese results indicate that IL-1β may be involved in the pulpal inflammatory and healing processes by inducing MCP-1 expression and secretion. These events are related to differential activation of TAK1, PI3K/Akt and MEK/ERK 1/2 signaling and COX activation. These results are important for future pharmacologic intervention of pulpal inflammatory diseases.

Abstract P023: Toll-like Receptor 4 Deficiency Reduces Macrophage Mediated Renal Injury in Ang-II Induced Hypertension

Infiltration of macrophages has been amply demonstrated in the kidney of humans and experimental models of hypertension however, its role in tissue injury and repair remains unclear. Furthermore, recent studies implicate upregulation of toll-like receptor 4 (TLR4) and inflammation in hypertension and vascular remodeling. Previously, we observed that C3H/HeJ mice lacking functional TLR4 were hyporesponsive to Ang-II induced hypertension and renal damage. In this study, we hypothesized that TLR4 deficiency inhibits macrophage mediated inflammation and TGF-β-induced fibroblast accumulation to reduce renovascular injury and fibrosis. Male C3H/HeOuJ (functional TLR4) and C3H/HeJ mice (dysfunctional TLR4) aged 8-10 weeks underwent subcutaneous osmotic pump insertion for infusion of Angiotensin-II (Ang-II) at 1000 ng/kg/min or vehicle (0.9% saline) for 4 weeks. Blood pressure (BP) was measured twice weekly by non-invasive tail-cuff plethysmography. Ang-II treatment increased mean BP in both groups but to a greater degree in C3H/HeOuJ mice compared to C3H/HeJ (138.6± 6.3 vs. 103.4±7.2 mm/Hg). Plasma creatinine in saline treated C3H/HeOuJ and C3H/HeJ mice was normal (0.37±0.026 and 0.32±0.023 mg/dL respectively) in contrast to ang-II treated mice (1.34±0.075 vs. 0.78±0.052 mg/dL). Flow cytometry revealed increased F4/80+ and CD40+ cells indicating pro-inflammatory M1 macrophage in Ang-II treated C3H/HeOuJ compared to C3H/HeJ mice. Similarly, Ang-II increased the mRNA and protein expression of CD40, MCP-1, TNF-α and TGF-β in C3H/HeOuJ compared to C3H/HeJ mice. Immunostaining revealed increased colocalization of CD45 and procollagen-1 in renal sections of C3H/HeOuJ mice suggesting increased accumulation of myeloid fibroblasts than in C3H/HeJ mice. The expression of α-smooth muscle actin an indicator of myofibroblast and extracellular matrix proteins, collagen type I and fibronectin (Western blot and immunostaining) was increased in C3H/HeOuJ compared to C3H/HeJ mice. In conclusion, our data suggests that in C3H/HeJ mice, deficiency of functional TLR4 reduces macrophage activation and inflammation which in turn decreases TGF-β-induced renal fibrotic response in Ang-II induced hypertension.

Engineered composite fascia for stem cell therapy in tissue repair applications

A critical challenge in tissue regeneration is to develop constructs that effectively integrate with the host tissue. Here, we describe a composite, laser micromachined, collagen–alginate construct containing human mesenchymal stem cells (hMSCs) for tissue repair applications. Collagen type I was fashioned into laminated collagen sheets to form a mechanically robust fascia that was subsequently laser micropatterned with pores of defined dimension and spatial distribution as a means to modulate mechanical behavior and promote tissue integration. Significantly, laser micromachined patterned constructs displayed both substantially greater compliance and suture retention strength than non-patterned constructs. hMSCs were loaded in an RGD-functionalized alginate gel modified to degrade in vivo. Over a 7day observation period in vitro, high cell viability was observed with constant levels of VEGF, PDGF-β and MCP-1 protein expression. In a full thickness abdominal wall defect model, the composite construct prevented hernia recurrence in Wistar rats over an 8-week period with de novo tissue and vascular network formation and the absence of adhesions to underlying abdominal viscera. As compared to acellular constructs, constructs containing hMSCs displayed greater integration strength (cell seeded: 0.92±0.19N/mm vs. acellular: 0.59±0.25N/mm, p=0.01), increased vascularization (cell seeded: 2.7–2.1/hpf vs. acellular: 1.7–2.1/hpf, p<0.03), and increased infiltration of macrophages (cell seeded: 2021–3630μm2/hpf vs. acellular: 1570–2530μm2/hpf, p<0.05). A decrease in the ratio of M1 macrophages to total macrophages was also observed in hMSC-populated samples. Laser micromachined collagen–alginate composites containing hMSCs can be used to bridge soft tissue defects with the capacity for enhanced tissue repair and integration. Statement of SignificanceEffective restoration of large soft tissue defects caused by trauma or treatment complications represents a critical challenge in the clinic. In this study, a novel composite construct was engineered and evaluated for stem cell delivery and tissue repair. Laser micromachining was used to fabricate patterned, microporous constructs designed with pores of defined size and distribution as a means to tune mechanical responses, accommodate and protect incorporated cells, and enhance tissue integration. The construct was embedded within an engineered alginate gel containing hMSCs. Upon repair of a full thickness abdominal wall defect in a rat model, the composite construct modulated host innate immunity towards a reparative phenotypic response, promoted neovascularization and associated matrix production, and increased the strength of tissue integration.

Abstract 2359: Adrenergic regulation of MCP-1 leads to enhanced macrophage recruitment and ovarian carcinoma growth

Abstract Increased adrenergic signaling is known to promote tumor progression, but the underlying mechanisms remain poorly understood. Tumor associated macrophages (TAMs) are key components of the tumor microenvironment that contribute to pro-inflammatory processes and tumor growth. Recently, it has been reported that patients with higher levels of adrenergic signaling have higher counts of MMP-9-producing TAMs. Here, we examined factors responsible for stress-mediated effects on monocyte/macrophage recruitment into the tumor microenvironment and the resultant effects on tumor growth. Conditioned media from norepinephrine- or epinephrine-treated ADRB positive SKOV3ip1 ovarian cancer cells had significantly increased levels of pro-inflammatory cytokines, such as MCP1, fractalkine, IL6, IL8 and VEGF. MCP1, a key modulator of monocyte/macrophage recruitment, gene and protein levels were significantly increased in vitro after catecholamine exposure, which was mediated by cAMP and PKA, while this effect was abrogated by a beta-blocker. SKOV3ip1 tumor samples from mice, subjected to daily restraint stress, had elevated MCP1 gene and protein expression levels, increased CD14+ cells, and increased infiltration of CD68+ cells. Both, propranolol, a non-specific beta-blocker, and hMCP1 siRNA packaged in DOPC nanoparticles significantly abrogated daily restraint stress-induced tumor growth and inhibited infiltration of CD68+ and F4/80+ cells. Of patient samples evaluated (n = 462), 28% had elevated absolute peripheral blood monocyte (&amp;gt;0.7 × 106/μL) and 43% had high peripheral blood monocytes when measured as the percent of white blood cell content (&amp;gt;8%). Elevated peripheral blood monocytes and increased CD68+ cells (&amp;gt;15/hpf) in samples from ovarian cancer patients were significantly associated with advanced disease and worse overall survival. In summary, increased adrenergic signaling is associated with enhanced macrophage infiltration mediated by tumor cell-derived MCP1 production. Citation Format: Guillermo N. Armaiz-Pena, Vianey Gonzalez-Villasana, Archana S. Nagaraja, Cristian Rodriguez-Aguayo, Piotr Dorniak, Rebecca Previs, Nouara Sadaoui, Rebecca Stone, Koji Matsuo, Heather J. Dalton, Susan K. Lutgendorf, Anil K. Sood, Gabriel Lopez-Berestein. Adrenergic regulation of MCP-1 leads to enhanced macrophage recruitment and ovarian carcinoma growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2359. doi:10.1158/1538-7445.AM2015-2359

Upregulated expression of monocyte chemoattractant protein‐1 in human periodontal ligament cells induced by interleukin‐1β

Root resorption during orthodontic treatment is a complex and sterile inflammatory process, characterized by the recruitment of mononuclear cells in the local periodontal ligament. This study aimed to investigate whether interleukin (IL)-1β could induce the migration of monocytes through upregulating monocyte chemoattractant protein (MCP)-1 expression in human periodontal ligament cells. Human periodontal ligament cells were cultured in medium containing various IL-1β concentrations. After 24hours of incubation, the messenger RNA (mRNA) and MCP-1 protein in periodontal ligament cells were detected by reverse transcriptase-polymerase chain reaction and Western blot analysis, respectively. The effect of supernatants of periodontal ligament cells on THP-1 cells was analysed via migration assay. IL-1β (10ng/mL and 25ng/mL) increased the expression of MCP-1 mRNA and protein in periodontal ligament cells (p<0.05). In addition, the supernatants from the IL-1β-treated periodontal ligament cells increased the migratory response of THP-1 cells (p<0.05), which could be blocked by the anti-MCP-1 antibody. IL-1β had the potential to induce the migratory response of monocytes via upregulation of the expression of MCP-1 in human periodontal ligament cells and could contribute to orthodontic root resorption.

Neutralizing Anti-IL20 Antibody Treatment Significantly Modulates Low Grade Inflammation without Affecting HbA1c in Type 2 Diabetic db/db Mice.

Low grade inflammation is present in pre-clinical and human type 2 diabetes. In this process, several cytokines like IL-1β and inflammatory cells like macrophages are activated and demonstrated to participate to the disease initiation and progression. IL-20 is a cytokine known to play non-redundant roles in progression of several inflammatory diseases. To address the therapeutic effect of inhibiting the IL-20 pathway in diabetes, diabetic db/db mice were treated with neutralizing anti-IL20 antibodies in vivo and both metabolic and inflammatory parameters were followed. Diabetic islets expressed the IL-20 cytokine and all IL-20 receptor components in elevated levels compared to resting non-diabetic islets. Islets were responsive to ex vivo IL-20 stimulation measured as SOCS induction and KC and IL-6 production. Neutralizing anti-IL20 treatment in vivo had no effect on HbA1c or weight although the slope of blood glucose increase was lowered. In contrast, anti-IL20 treatment significantly reduced the systemic low-grade inflammation and modulated the local pancreatic immunity. Significant reduction of the systemic IL-1β and MCP-1 was demonstrated upon anti-IL20 treatment which was orchestrated with a reduced RANTES, IL-16 and IL-2 but increased TIMP-1, MCP-1 and IL-6 protein expression locally in the pancreas. Interestingly, anti-IL20 treatment induced an expansion of the myeloid suppressor CD11bGr1int macrophage while reducing the number of CD8 T cells. Taken together, anti-IL20 treatment showed moderate effects on metabolic parameters, but significantly altered the low grade local and systemic inflammation. Hence, future combination therapies with anti-IL20 may provide beneficial therapeutic effects in type 2 diabetes through a reduction of inflammation.

Pure peptides from amaranth (Amaranthus hypochondriacus) proteins inhibit LOX-1 receptor and cellular markers associated with atherosclerosis development in vitro

The objectives were to evaluate and to compare the effect of pure peptides from amaranth proteins on markers that promote atherosclerosis, in vitro. Amaranth pure peptides with potential anti-atherosclerotic effect, HGSEPFGPR, RPRYPWRYT and RDGPFPWPWYSH, were studied using lipopolysaccharide-induced THP-1 human macrophage-like cells. Lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) expression was reduced by peptides HGSEPFGPR (83%), RDGPFPWPWYSH (49%) and RPRYPWRYT (63%); while intracellular adhesion molecule-1 (ICAM-1) was reduced by peptides RDGPFPWPWYSH (27%) and RPRYPWRYT (39%); and matrix metalloproteinase-9 (MMP-9) expression was reduced by 52% (HGSEPFGPR), 41% (RDGPFPWPWYSH) and 29% (RPRYPWRYT). Using confocal microscopy, peptide HGSEPFGPR reduced LOX-1, ICAM-1 and MMP-9 expression by 74%, 59% and 80%, respectively. RPRYPWRYT reduced LOX-1, ICAM-1 and MMP-9 expression by 48%, 49% and 71%, respectively. RPRYPWRYT reduced MCP-1 and TGF-β cellular protein expression (74% and 78%, respectively); 89% (IL-6 and IL-1α); 83% (IFN-γ); 87% and 58% (TNF-α and TNF-β). HGSEPFGPR showed significant reduction of 27%, 31%, 41%, 57%, and 61% for GRO-α, MCP-1, IL-6, IL-1α, and RANTES, respectively. HGSEPFGPR showed better interactions with LOX-1 crystal structure than RPRYPWRYT. Amaranth pure peptides reduced the expression of proteins associated with LOX-1 signaling pathway, in vitro.

Chronic Intermittent Hypobaric Hypoxia Ameliorated Inflammation of Skeletal Muscle via Inhibited NF-kappa B in Rats with Metabolic Syndrome

This study was aimed to confirm the hypothesis that Chronic intermittent hypobaric hypoxia (CIHH) could ameliorate inflammation of skeletal muscle in rats with metabolic syndrome (MS). Sprague-Dawley rats (male, 160-180 g) were randomly divided into 4 groups: control group (CON), fructose group (FRUC, 10% fructose in drinking water for 6 weeks), CIHH group (simulated 5,000 m altitude, 6 h per day for 6 weeks), and CIHH plus fructose groups (CIHH-F). Histopathology of skeletal muscle, systolic arterial pressure (SAP), blood biochemical, protein expression of TNFα, MCP-1 and NF-kappa B were measured. The SAP, body weight, and the plasma contents of glucose, insulin, insulin C peptide, triglyceride and total cholesterol were elevated in FRUC rats but not in CIHH-F rats. Meanwhile, the inflammatory response of skeletal muscle was presented in FRUC rats but not in CIHH-F rats, which was demonstrated by the infiltration of circulating mononuclear cells and the up-regulation of TNFα and MCP-1. Finally the protein expression of NF-kappa B in skeletal muscle was correspondingly up-regulated in FRUC rats, but such upregulation was not observed in CIHH-F rats. Our results suggest that CIHH prevents the inflammation of skeletal muscle in rats with MS via inhibiting NF-kappa B signaling. CIHH might be the new strategy for prevention and therapy of MS.

Simulated microgravity promotes monocyte adhesion to rat aortic endothelium via nuclear factor-κB activation.

Microgravity-induced vascular remodelling may play an important role in post-spaceflight orthostatic intolerance. In this study, we aimed to investigate the effects of simulated microgravity on monocyte adhesion to aortic endothelium in hindlimb unweighted rats and to elucidate the underlying mechanisms associated with this event. Sprague-Dawley rats were subjected to 4-week hindlimb unweighting to simulate microgravity. The recruitment of monocytes to the abdominal aorta was investigated by en face immunofluorescence staining and monocyte binding assays. The expression of the adhesion molecules E-selectin and vascular cell adhesion molecule-1 as well as the cytokine monocyte chemoattractant protein (MCP)-1 was evaluated by immunohistochemical staining, western blot, and quantitative reverse transcription polymerase chain reaction analyses. Additionally, nuclear factor-κB (NF-κB) activation and the messenger RNA expression levels of E-selectin, vascular cell adhesion molecule-1, and MCP-1 were assessed with the administration of an NF-κB inhibitor, pyrrolidine dithiocarbamate. Results showed that simulated microgravity significantly increased monocyte recruitment to the aortic endothelium, protein expression of E-selectin and MCP-1, and NF-κB activation in the abdominal aorta of rats. Pyrrolidine dithiocarbamate treatment not only significantly inhibited NF-κB activity but also reduced the messenger RNA levels of E-selectin, vascular cell adhesion molecule-1, and MCP-1 as well as monocyte recruitment in the abdominal aorta of hindlimb unweighted rats. These results suggest that simulated microgravity increases monocyte adhesion to rat aortic endothelium via the NF-κB-mediated expression of the adhesion molecule E-selectin and the cytokine MCP-1. Therefore, an NF-κB-mediated inflammatory response may be one of the cellular mechanisms responsible for arterial remodelling during exposure to microgravity.

InactivationofAdiposeAngiotensinogen Reduces inflammatory Adipokines and Adipose Tissue Macrophages

Adiposerennin-angiotensin systemhasbeen linked to obesity and metabolic syndrome. Previously we showed that overexpression of Angiotensinogen(Agt) in adipose tissue increased insulin resistance, adipose and systemic inflammation in mice fed a low fat(LF)diet. To further dissect the direct role of adipose Agt in metabolic disorders,we have created an adipose specific Agt knockout (Agt-KO)mice using the Cre-LoxPsystem.Agt-KO and control(WT) littermates were fed either a low-fat (LF) or high-fat (HF) diet to assess metabolic changes. Surprisingly, most metabolic parameters (body weight and fat mass, glucose and insulin tolerance tests) were comparable between the WT and Agt-KO littermates in both diets. Analyses of adipose tissue gene expression indicated shifts in angiogenesis and insulin signaling in the Agt-KO mice fed LF diets when compared to the WT mice. MCP1 gene expression was also downregulated in adipose tissue of the Agt-KO vs. WT littermates. These changes correlated with MCP-1 and Leptin protein expression, both of which were downregulated in the Agt-KO. Moreover,targeted inactivation of adipose Agt reduced total macrophage infiltration in both the LF and HF fed Agt-KO mice. This was in contrast to the total adipocyte area,which did not change between the two genotypes. In conclusion, despite the lack of an obvious phenotype in adipose Agt deficient mice, cellular and molecular changes observed are consistent with previously reported functions of RAS in insulin resistance, and inflammation.

Impaired cytokine expression, neutrophil infiltration and bacterial clearance in response to urinary tract infection in diabetic mice.

Diabetic patients have increased susceptibility to infections, and urinary tract infections (UTI) are the most common type in women with diabetes mellitus. Knowledge of bacterial clearance effectiveness following UTI in diabetics is sparse. In this study, the effects of diabetes on bacterial clearance efficiency and components of the innate immune system in response to UTI in a murine model were investigated. Streptozotocin-induced diabetic and control female C57BL/6J mice were infected with uropathogenic Escherichia coli, and bacterial load, expression of chemokines, and neutrophil infiltration in the bladder over time were investigated. Expression levels of histone deacetylases were also measured to address a potential mechanism underlying the phenotype. Bacterial clearance during UTI was significantly prolonged in diabetic mice relative to controls. Neutrophil infiltration in bladder tissue and urine, and both mRNA and protein expression of chemokines MIP-2, KC, MCP-1 and IL-6 in bladder tissue were diminished at early time points after infection in diabetic mice relative to controls. In addition, mRNA levels of histone deacetylases 1-5 were increased in diabetic mice. This is the first study to show an association of impaired bacterial clearance in diabetic mice with suppression of UTI-induced chemokine expression and neutrophil infiltration in the bladder.