Monocyte Chemotactic Protein-1 mRNA Research Articles

PD9-02 TRANSGENIC MICE THAT SECRETE MCP-1 BY THE UROTHELIUM DEMONSTRATE BLADDER HYPERSENSITIVITY, PELVIC PAIN AND URINARY SYMPTOMS

You have accessJournal of UrologyInfections/Inflammation of the Genitourinary Tract: Interstitial Cystitis1 Apr 2014PD9-02 TRANSGENIC MICE THAT SECRETE MCP-1 BY THE UROTHELIUM DEMONSTRATE BLADDER HYPERSENSITIVITY, PELVIC PAIN AND URINARY SYMPTOMS Yi Luo, Liwei Liu, Xu Wang, Michael O'Donnell, and Karl Kreder Yi LuoYi Luo More articles by this author , Liwei LiuLiwei Liu More articles by this author , Xu WangXu Wang More articles by this author , Michael O'DonnellMichael O'Donnell More articles by this author , and Karl KrederKarl Kreder More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.784AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Excessive production of monocyte chemotactic protein-1 (MCP-1) has been observed in various inflammatory, chronic pain, and bladder overactive conditions. We have observed that over half of patients with interstitial cystitis/bladder pain syndrome (IC/BPS) express elevated MCP-1 in the urine. This study was to develop a mouse model that secretes MCP-1 by the urothelium to facilitate the study of IC/BPS in humans. METHODS A 4.9 Kb transgene consisting of the uroplakin II gene promoter and mouse MCP-1 coding sequence with a secretory element was constructed and microinjected for developing a MCP-1 secreting transgenic mouse line (URO-MCP-1). Mice were screened by tail genotyping and backcrossed onto the C57BL/6 genetic background. RT-PCR was used to assess MCP-1 mRNAs in the bladder and ELISA used to assess MCP-1 protein in the urine. To induce bladder inflammation, lipopolysaccharide (LPS) was administered intravesically. Bladder inflammation was analyzed by histological hematoxylin and eosin (H&E). Von Frey filament stimulation was used to assess pelvic pain and micturition cages used to assess voiding dysfunction. Cadi-05, a novel Toll-like receptor modulator, was intradermally administered once daily for total three doses starting one day before cystitis induction up to day 1. The bladders were analyzed by histological H&E staining at day 2. RESULTS URO-MCP-1 mice express MCP-1 mRNA in the bladder but not in other organs. Urine contains ∼1,500 pg/ml of MCP-1. While the bladders of URO-MCP-1 mice were normal in the unmanipulated state, they exhibited a much lower threshold trigger for producing exaggerated responses to otherwise sub-noxious inflammatory stimuli such as LPS. Compared to control C57BL/6 mice, URO-MCP-1 mice exhibited clear histological bladder inflammation with intensive edema and cellular infiltration 24 hours after intravesical instillation of even a single low does of LPS (1 μg in 100 μl). The bladder inflammation peaks at days 1-3 and lasts 5-7 days. URO-MCP-1 mice exhibited pelvic pain and increased urinary frequency and decreased urine output per void after cystitis induction (p<0.05). Intradermal Cadi-05 treatment significantly reduced bladder histopathology (p<0.01). CONCLUSIONS The URO-MCP-1 model exhibits key symptomatology of IC/BPS and is responsive to intradermal Cadi-05 therapy. This model offers a unique translational opportunity for investigation of the pathological mechanisms and therapeutic development for the human disease. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e212 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Yi Luo More articles by this author Liwei Liu More articles by this author Xu Wang More articles by this author Michael O'Donnell More articles by this author Karl Kreder More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

PD9-03 INFLAMMATORY BIOMARKERS ARE ASSOCIATED WITH CO-MORBID PAIN CONDITIONS IN UCPPS PATIENTS: A MAPP STUDY

INTRODUCTION AND OBJECTIVES: Excessive production of monocyte chemotactic protein-1 (MCP-1) has been observed in various inflammatory, chronic pain, and bladder overactive conditions. We have observed that over half of patients with interstitial cystitis/bladder pain syndrome (IC/BPS) express elevated MCP-1 in the urine. This study was to develop a mouse model that secretes MCP-1 by the urothelium to facilitate the study of IC/BPS in humans. METHODS: A 4.9 Kb transgene consisting of the uroplakin II gene promoter and mouse MCP-1 coding sequence with a secretory element was constructed and microinjected for developing a MCP-1 secreting transgenic mouse line (URO-MCP-1). Mice were screened by tail genotyping and backcrossed onto the C57BL/6 genetic background. RT-PCR was used to assess MCP-1 mRNAs in the bladder and ELISA used to assess MCP-1 protein in the urine. To induce bladder inflammation, lipopolysaccharide (LPS) was administered intravesically. Bladder inflammation was analyzed by histological hematoxylin and eosin (H&E). Von Frey filament stimulation was used to assess pelvic pain and micturition cages used to assess voiding dysfunction. Cadi-05, a novel Toll-like receptor modulator, was intradermally administered once daily for total three doses starting one day before cystitis induction up to day 1. The bladders were analyzed by histological H&E staining at day 2. RESULTS: URO-MCP-1 mice express MCP-1 mRNA in the bladder but not in other organs. Urine contains w1,500 pg/ml of MCP-1. While the bladders of URO-MCP-1 mice were normal in the unmanipulated state, they exhibited a much lower threshold trigger for producing exaggerated responses to otherwise sub-noxious inflammatory stimuli such as LPS. Compared to control C57BL/6 mice, URO-MCP-1 mice exhibited clear histological bladder inflammation with intensive edema and cellular infiltration 24 hours after intravesical instillation of even a single low does of LPS (1 mg in 100 ml). The bladder inflammation peaks at days 1-3 and lasts 5-7 days. UROMCP-1 mice exhibited pelvic pain and increased urinary frequency and decreased urine output per void after cystitis induction (p<0.05). Intradermal Cadi-05 treatment significantly reduced bladder histopathology (p<0.01). CONCLUSIONS: The URO-MCP-1 model exhibits key symptomatology of IC/BPS and is responsive to intradermal Cadi-05 therapy. This model offers a unique translational opportunity for investigation of the pathological mechanisms and therapeutic development for the human disease.

Tauroursodeoxycholic acid reduces glial cell activation in an animal model of acute neuroinflammation

BackgroundBile acids are steroid acids found predominantly in the bile of mammals. The bile acid conjugate tauroursodeoxycholic acid (TUDCA) is a neuroprotective agent in different animal models of stroke and neurological diseases. However, the anti-inflammatory properties of TUDCA in the central nervous system (CNS) remain unknown.MethodsThe acute neuroinflammation model of intracerebroventricular (icv) injection with bacterial lipopolysaccharide (LPS) in C57BL/6 adult mice was used herein. Immunoreactivity against Iba-1, GFAP, and VCAM-1 was measured in coronal sections in the mice hippocampus. Primary cultures of microglial cells and astrocytes were obtained from neonatal Wistar rats. Glial cells were treated with proinflammatory stimuli to determine the effect of TUDCA on nitrite production and activation of inducible enzyme nitric oxide synthase (iNOS) and NFκB luciferase reporters. We studied the effect of TUDCA on transcriptional induction of iNOS and monocyte chemotactic protein-1 (MCP-1) mRNA as well as induction of protein expression and phosphorylation of different proteins from the NFκB pathway.ResultsTUDCA specifically reduces microglial reactivity in the hippocampus of mice treated by icv injection of LPS. TUDCA treatment reduced the production of nitrites by microglial cells and astrocytes induced by proinflammatory stimuli that led to transcriptional and translational diminution of the iNOS. This effect might be due to inhibition of the NFκB pathway, activated by proinflammatory stimuli. TUDCA decreased in vitro microglial migration induced by both IFN-γ and astrocytes treated with LPS plus IFN-γ. TUDCA inhibition of MCP-1 expression induced by proinflammatory stimuli could be in part responsible for this effect. VCAM-1 inmunoreactivity in the hippocampus of animals treated by icv LPS was reduced by TUDCA treatment, compared to animals treated with LPS alone.ConclusionsWe show a triple anti-inflammatory effect of TUDCA on glial cells: i) reduced glial cell activation, ii) reduced microglial cell migratory capacity, and iii) reduced expression of chemoattractants (e.g., MCP-1) and vascular adhesion proteins (e.g., VCAM-1) required for microglial migration and blood monocyte invasion to the CNS inflammation site. Our results present a novel TUDCA anti-inflammatory mechanism, with therapeutic implications for inflammatory CNS diseases.

Synchronous regulation of the determinants of endometrial receptivity to interleukin 1 at key stages of early embryo implantation in vivo

To investigate the expression kinetics of interleukin-1 receptors (IL-1R), receptor antagonist (IL-1RN), and monocyte chemotactic protein 1 (MCP-1) throughout early gestation in mice. Assessment of IL-1R, IL-1RN, and MCP-1 throughout early pregnancy. Reproduction laboratory. B6C3F1 female mice bred with fertile males of the same strain. Collection of endometrial tissue at necropsy from nonimplanted and implanted sites. IL-1R, IL-1RN, and MCP-1 mRNA expression by quantitative reverse-transcription polymerase chain reaction and protein expression by enzyme-linked immunosorbent assay and immunohistochemistry. The expression of the signaling IL-1R1 significantly increased in the first 2 days of gestation, which corresponded to the inflammatory-like period triggered by the seminal fluid, before increasing again at the implantation window and lasting throughout embryo implantation. The expression of inhibitory IL-1R2 and IL-1RN concomitantly increased during gestational days 1-2 but remained low, particularly within the embryo implantation sites and throughout the implantation period. The expression of MCP-1 significantly increased only at the embryo implantation sites and showed a significant positive correlation with IL-1R1 expression. Our data identified for the first time synchronous changes in endometrial IL-1R throughout early gestation in vivo and point to a deep modulation of endometrial receptivity to IL-1 by embryo-driven signals. This may play a key role in the creation of a receptive phenotype in the maternal endometrium and represent a key mechanism underlying embryo implantation.

Lipoxin A4 attenuation of endothelial inflammation response mimicking pancreatitis-induced lung injury

Lipoxins (LXs) and their analogues are known to display potent anti-inflammatory actions. Previously, we reported that lipoxin A4 (LXA4) possessed powerful anti-inflammatory properties in acute pancreatitis in rats and that it may ameliorate the concomitant acute lung injury by reducing cytokine generation and inhibiting neutrophil activation. Considering that the vascular endothelium plays an important role during adherence, migration and activation of leukocytes, the present study was designed to investigate the effects of LXA4 on the inflammatory response induced by tumor necrosis factor α (TNF-α) in human pulmonary microvascular endothelial cells (HPMECs) and explore the potential mechanisms involved in these processes. We found that LXA4 markedly down-regulated the expression of monocyte chemotactic protein-1 (MCP-1), E-selectin, and interleukin-6 (IL-6) mRNA, as well as intercellular adhesion molecule-1 (ICAM-1) in TNF-α-exposed HPMECs. Moreover, LXA4 inhibited the phosphorylation and nuclear translocation of nuclear factor-κB/p65 (NF-κB/p65) and phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) in HPMECs following TNF-α stimulation. Heme oxygenase-1 (HO-1), a cytoprotective enzyme, was up-regulated by LXA4 in both non- and TNF-α-stimulated HPMECs. In conclusion, the protective effects of LXA4 to ALI may be executed through inhibition inflammation pathways of NF-κB and p38 MAPK and up-regulation of cytoprotective HO-1.

Effects of cyclic stretch on expression of cytokines and intercellular adhesion molecule-1 in human pulmonary artery endothelial cell

To observe the effects of cyclic stretch on expression of cytokines and adhesion molecules in human pulmonary artery endothelial cells (HPAECs), herein to provide a theoretical basis to ventilator-induced lung injury (VILI). HPAECs were subjected to cyclic stretch by the Flexcell FX-5000T system at 0.5 Hz of 10% or 20% elongation for 3, 6, 12, 24 hours respectively. The mRNA and protein expression of interleukin (IL-6, IL-8), monocyte chemotactic protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) was determined by fluorescent quantitation reverse transcription-polymerase chain reaction (qRT-PCR), enzyme linked immunosorbent assay (ELISA) or Western blotting. Increasing the stretch force, the mRNA and protein expression of IL-8, MCP-1, ICAM-1 were up regulated with increasing stretch time. Compared with the control (set 1), after 20% cyclic stretch for 24 hours, IL-8 mRNA expression was up regulated to 1.58±0.10, MCP-1 mRNA expression was up regulated to 2.85±0.52, and ICAM-1 mRNA expression was up regulated to 1.90±0.14 (all P<0.05). Compared with control group, after 20% cyclic stretch for 24 hours, the protein expression of IL-8 and MCP-1 in HPAEC was significantly increased (IL-8: 3401.08±439.60 ng/L vs. 1422.60±66.98 ng/L, MCP-1: 1117.64±237.54 ng/L vs. 307.88±80.84 ng/L, both P<0.05), ICAM-1 protein expression was up regulated to 2.15±0.40 (P<0.05), while the expression of IL-6 mRNA and protein had no statistic difference compared with control group. Cyclic stretch enhanced the expression of IL-8, MCP-1 and ICAM-1 in an intensity-dependent fashion, so it may be involved in the pathogenesis of lung injury induced by mechanical ventilation.

Mo1898 Macrophage Related Inflammation and Phenotype Modulation in Barrett's Esophagus

Central obesity, independent of gastroesophageal reflux, is a risk factor for the development of Barrett's esophagus (BE). Low grade chronic inflammation mediated by the innate immune system is a well-recognized feature of organs affected by obesity, such as adipose tissue and liver. Plasma free fatty acids (FFA) are elevated in obese patients. Recent studies have shown that macrophage inflammatory responses can be modulated by FFAs. Therefore, we hypothesized that macrophage activation plays a pathogenic role in Barrett's esophagus. Our objective was to profile proand anti-inflammatory macrophage markers in esophageal mucosa from BE patients in regions of intestinal metaplasia and corresponding squamous epithelium and assess the influence of FFAs on macrophage activation in vitro. Methods: Patients with BE and matched controls without endoscopic evidence of BE were included. Tissue obtained by esophageal biopsy at esophagogastroscopy was used to extract total RNA. Complementary DNA was generated and tissue expression of genes of interest determined by quantitative polymerase chain reaction using probe-based Taqman chemistry and commercially available function-tested RealTime ready assays. Human monocyte cells, THP.1, were differentiated into macrophages and treated with FFAs. Results: BE samples showed significant macrophage accumulation compared to controls as measured by relative CD68 mRNA expression, which was 1.5 fold higher in cases (p,0.05). This was confirmed by immunohistochemistry for macrophage markers, which showed a statistically significant predominance of pro-inflammatory macrophages in BE tissue. We next profiled the expression of proinflammatory markers: interleukin-1β (IL-1β) and monocyte chemotactic protein-1 (MCP1) and anti-inflammatory markers: interleukin 10 (IL10), CD206 and arginase-1 in these samples. This revealed a trend toward a proinflammatory signature in BE tissue (higher MCP1 and lower arginase-1 mRNA abundance) compared to squamous tissue from controls and BE subjects. Further, in complementary in vitro experiments using a human macrophage cell line, we demonstrated that the saturated FFA, palmitic acid (PA), significantly increased TNF alpha, MCP-1, and IL-1βmRNA expression, consistent with pro-inflammatory polarization and the polyunsaturated FFA, docosahexaenoic acid (DHA), increased the mRNA expression of CD206 and arginase-1 consistent with anti-inflammatory polarization (Figure

Effects of cordyceps acid and cordycepin on the inflammatory and fibrogenic response of hepatic stellate cells

To investigate the effects of cordyceps acid and cordycepin on the inflammatory phenotype and fibrogenic property of hepatic stellate cells (HSCs). An immortalized mouse HSC line (JS1) was stimulated with lippolysaccharide (LPS; 100 ng/ml) to induce an inflammatory response with or without co-administration of cordyceps acid or cordycepin in various concentrations (10, 50, or 200 mumol/L). Effects of the treatments on the chemokine monocyte chemotactic protein-1 (MCP-1) mRNA expression in the cells and the protein secretion in the cell culture supernatants were determined by reverse transcription and real-time quantitative PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. In addition, JS1 cells were treated with transforming growth factor-b1 (TGFb1; 10 ng/ml) to induce a fibrogenic response with or without co-administration of cordyceps acid or cordycepin in various concentrations (10, 50, or 200 mumol/L). Effects on the expression of fibrogenic proteins including collagen type I and a-smooth muscle actin (a-SMA), were investigated by Western blot. High-concentration (200 mumol/L) treatments of both cordyceps acid and cordycepin significantly inhibited the LPS-induced up-regulation of MCP-1 transcription and secretion (mRNA: 2.07 +/- 0.29 vs. 3.35 +/- 0.26, t = 15.90 and 1.15 +/- 0.23 vs. 4.17 +/- 0.61, t = 8.93; protein: 1.88 +/- 0.06 vs. 2.33 +/- 0.06, t = 10.39 and 1.47 +/- 0.25 vs. 1.97 +/- 0.04, t = 4.60; all P less than 0.05). All concentrations of cordyceps acid and cordycepin inhibited the TGFb1-induced up-regulation of collagen type I and a-SMA protein expression. However, the effects were more robust with the 200 mumol/L concentrations (P less than 0.05). Cordyceps acid and cordycepin ameliorate the LPS-induced inflammatory phenotype and TGFb1-induced fibrogenic response of cultured HSCs. These effects may contribute significantly to the drugs' therapeutic mechanisms to inhibit and resolve liver fibrosis.

Sevoflurane suppresses tumour necrosis factor-α-induced inflammatory responses in small airway epithelial cells after anoxia/reoxygenation

Lung ischaemia-reperfusion (I/R) injury is correlated with poor clinical outcome. The inflammatory cytokines interleukin (IL)-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) are produced by pulmonary epithelial cells during lung transplantation and are considered to be involved in I/R injury. The volatile anaesthetic sevoflurane has been shown to exert a protective effect on I/R injury in various organs. We investigated the effect of sevoflurane on the inflammatory functions of pulmonary epithelial cells in vitro. Human normal small airway epithelial cells (SAEC) were incubated under anoxic conditions for 24 h with or without sevoflurane and then stimulated with tumour necrosis factor (TNF)-α under hyperoxic conditions for 5 h with or without sevoflurane. After incubation, IL-6, IL-8, and MCP-1 mRNA expression was analysed by quantitative real-time RT-PCR. The production of IL-6, IL-8, and MCP-1 was assayed by enzyme-linked immunosorbent assay, the effects of sevoflurane on inflammatory gene expression were examined by DNA microarray analysis, and the effects of sevoflurane on NF-κB-mediated inflammatory cytokine production were examined by immunoblotting. Sevoflurane suppressed TNF-α-induced IL-6, IL-8, and MCP-1 gene expression and the production of IL-6 and IL-8 in SAEC under anoxia/reoxygenation conditions. DNA microarray analysis indicated that sevoflurane modulated NF-κB-related gene expression. Sevoflurane significantly inhibited TNF-α-induced translocation of p65 NF-κB into the nucleus. Sevoflurane enhanced TNF-α-induced gene expression of inhibitor κB (IκB) but not of NF-κB. Sevoflurane suppressed the NF-κB-mediated production of pulmonary epithelial cell-derived inflammatory cytokines, including IL-6 and IL-8, which are capable of causing I/R injury.

MCP-1 Expression Is Specifically Regulated During Activation of Skeletal Repair and Remodeling

Monocyte chemotactic protein-1 (MCP-1) belongs to the CC chemokine superfamily and plays a critical role in the recruitment and activation of leukocytes during acute inflammation. We hypothesize that MCP-1 is also an important chemokine that regulates the recruitment and activation of bone cells required for skeletal repair and remodeling. We used the ulnar stress fracture (SFx) model, which allows investigation of focal remodeling with a known time course and precise anatomical location. SFx were created in the right ulna of female Wistar rats using cyclic end loading. Unloaded animals were used as a control. Rats were killed 4h and 1, 4, 7, and 14days after loading (n=10/group); RNA was extracted and converted to cDNA for quantitative PCR analysis using TaqMan gene expression assays. Four hours after loading, MCP-1 gene expression was increased ~30-fold (P<0.001), remained elevated at 24h (~12-fold, P<0.001), then declined by day 14. Relative to the contralateral limb, expression of the receptors CCR1 and CCR2 increased over the 14days, being significant by 4days for CCR1 and 14days for CCR2 (P<0.05). Other inflammation-related chemokines (RANTES, MIP1a) were not increased at these early time points. Using in situ hybridization and immunohistochemistry in separate animal groups (n=5/group, control, days 1, 4, 7), MCP-1 mRNA and protein were localized in periosteal osteoblasts associated with woven bone formation at the fracture exit point but not in osteocytes adjacent to the SFx. These data support an important role for MCP-1 in the early phase of SFx repair and activated remodeling.

Immunomodulatory Effect of Chinese Herbal Medicine Formula Sheng-Fei-Yu-Chuan-Tang in Lipopolysaccharide-Induced Acute Lung Injury Mice

Traditional Chinese medicine formula Sheng-Fei-Yu-Chuan-Tang (SFYCT), consisting of 13 medicinal plants, was used to treat patients with lung diseases. This study investigated the immunoregulatory effect of SFYCT on intratracheal lipopolysaccharides- (LPS-) challenged acute lung injury (ALI) mice. SFYCT attenuated pulmonary edema, macrophages, and neutrophils infiltration in the airways. SFYCT decreased inflammatory cytokines, including tumor necrosis factor-α (TNFα), interleukin-1β, and interleukin-6 and inhibited nitric oxide (NO) production but increased anti-inflammatory cytokines, interleukin-4, and interleukin-10, in the bronchoalveolar lavage fluid of LPS-challenged mice. TNFα and monocyte chemotactic protein-1 mRNA expression in the lung of LPS-challenged mice as well as LPS-stimulated lung epithelial cell and macrophage were decreased by SFYCT treatment. SFYCT treatment also decreased the inducible nitric oxide synthase expression and phosphorylation of nuclear factor-κB (NF-κB) in the lung of mice and macrophage with LPS stimulation. SFYCT treatment dose dependently decreased the LPS-induced NO and reactive oxygen species generation in LPS-stimulated macrophage. In conclusion, SFYCT attenuated lung inflammation during LPS-induced ALI through decreasing inflammatory cytokines production while increasing anti-inflammatory cytokines production. The immunoregulatory effect of SFYCT is related to inhibiting NF-κB phosphorylation.

Lysophosphatidic Acid Stimulates MCP-1 Secretion from C2C12 Myoblast

Chemokines are regulatory proteins that play an important role in muscle cell migration and proliferation. In this study, C2C12 cells treated with lysophosphatidic acid (LPA) showed an increase in endogenous monocyte chemotactic protein-1 (MCP-1) expression and secretion. LPA is a naturally occurring bioactive lysophospholipid with hormone- and growth-factor-like activities. LPA is produced by activated platelets, cytokine-stimulated leukocytes, and possibly by other cell types. However, the LPA analog cyclic phosphatidic acid (cPA) had no effect on the expression and secretion of MCP-1. LPA, although similar in structure to cPA, had potent inducing effects on MCP-1 expression in C2C12 cells. In this study, we showed that LPA enhanced MCP-1 mRNA expression and protein secretion in a dose-dependent manner. Taken together, these results suggest that LPA enhances MCP-1 secretion in C2C12 cells and thus may play an important role in cell proliferation.

Abstract 18473: ApoB-100 Related Peptide Immunization Confers Protection Against Angiotension II-induced Renal Inflammatory Responses

Background: T cells play an important role in angiotensin II (AngII)-induced hypertension and aneurysm formation. Inflammatory responses, typified by reactive oxygen species (ROS) and Interleukin-6 (IL-6), are involved in AngII-induced hypertension and renal damage. We recently demonstrated that immunization with apoB-100 related peptide p210 reduced atherosclerosis through T cell mediated immune response in apoE (-/-) mice. We also reported that immunization with p210 reduced mean arterial blood pressure (p210 = 124±4*, cBSA = 143±6, PBS = 139±3 mmHg. *p&lt;0.05 vs cBSA and PBS.) and aneurysm formation in AngII-infused apoE (-/-) mice. In this study, we assessed the effect of p210 vaccine on AngII-induced renal inflammatory responses in apoE (-/-) mice. Method and Results: ApoE (-/-) mice were immunized with p210/cBSA/Alum (p210; 100 μ g) at 7, 10, and 12 weeks of age. Mice receiving PBS or cBSA/Alum (cBSA) served as controls. At 10 weeks of age, mice were subcutaneously implanted with an osmotic pump which released AngII (1 mg/Kg/min), and were euthanized 4 weeks later. Serum creatinine level was significantly lower in p210 group (p210 = 0.7±0.1*, cBSA = 1.1±0.1, PBS = 1.4±0.1 mg/dL. N=12, 11, and 9 respectively; *p&lt;0.05 vs cBSA and PBS.). IL-6 and monocyte chemotactic protein-1 (MCP-1) mRNA expression in kidneys were significantly down-regulated in p210 group (IL-6: cBSA = 4.1±1.1, PBS = 3.9±1.0 -fold increase compared to p210; MCP-1: cBSA = 2.4±0.6, PBS = 2.2±0.4 -fold increase compared to p210, N=8). Renal glomerular superoxide production measured by in situ dihydroethidium was significantly decreased in p210 group (cBSA = 3.8±0.7, PBS = 3.9±0.6 -fold increase compared to p210, N=4.). The expression of NOX1, a component of NADPH oxidase, mRNA in kidneys was significantly down-regulated in p210 group (cBSA = 2.6±0.5, PBS = 2.8±0.4 -fold increase compared to p210, N=8.). Conclusion: Immunization with p210 significantly attenuated AngII-induced renal damage. These anti-hypertensive and renal protective effects were associated with down-regulation of IL-6 and MCP-1 expression, and ROS production mediated by NADPH oxidase in kidneys.

Inhibitory effect of CCR3 signal on alkali-induced corneal neovascularization.

To investigate the effect of CC chemokine receptor 3 (CCR3) signal on corneal neovascularization (CRNV) induced by alkali burn and to explore its mechanism. Specific pathogen-free male BALB/C mice (aged 6-8 weeks) were randomly divided into CCR3-antagonist treated group (experimental group) and control group. CRNV was induced by alkali burn in mice. The time kinetic CCR3 expression in injured corneas was examined by reverse transcription polymerase chain reaction (RT-PCR). CCR3-antagonist (SB-328437 at different concentration of 125µg/mL, 250µg/mL, and 500µg/mL) was locally administrated after alkali injury. The formation of CRNV was assessed by CD31 corneal whole mount staining at two weeks after injury. Monocyte chemotactic protein 1 (MCP-1), monocyte chemotactic protein 3 (MCP-3) expressions in the early phase after injury were quantified and compared by RT-PCR. Macrophage intracorneal accumulation in the early phase after injury was evaluated and compared by immunohistochemistry. Alkali injury induced the time kinetic intracorneal CCR3 expression. 500µg/mL of CCR3-antagonist treatment in the early phase but not the late phase resulted in significant impaired CRNV as compared to control group (P<0.05). CCR3-antagonist treatment in the early phase significantly reduced the intracorneal MCP-1 and MCP-3 enhancement compare to control group at day 2 and day 4 (P<0.05). Moreover, the number of intracorneal macrophage infiltration in the experimental group was reduced than those in control group at day 4 (P<0.05). CCR3 signal is involved in alkali-induced CRNV. CCR3-antagonist can inhibit alkali-induced CRNV by reducing the intracorneal MCP-1 and MCP-3 mRNA expression and the intracorneal macrophage infiltration.

Effect of secretin on preadipocyte, differentiating and mature adipocyte functions

Several gastrointestinal peptides are now recognized to have target functions beyond the intestinal wall, including effects on adipocytes. Secretin (SEC), one of the first identified, has not been evaluated in this context. Using cultured 3T3-L1 preadipocytes, adipocytes and primary rat adipocytes we evaluated the effect of SEC on cell proliferation, mitochondrial activity, differentiation, triglyceride (TG) synthesis, lipolysis as well expression of the SEC receptor (SCTR) in rodent and human adipose tissues. In preadipocytes, SEC significantly increased mitochondrial activity (115%; P<0.01), thymidine incorporation (149.7%; P<0.05) and C/EBPβ expression (123.4%; P<0.05). During standard differentiation, SCTR mRNA increased up to a maximum of ninefold (P<0.001). In human adipose tissue, SCTR correlated with body mass index and plasma insulin, and SCTR mRNA expression was also detected in rat adipose tissues. SEC supplementation during differentiation enhanced TG accumulation (+138%; P<0.01). In mature adipocytes, SEC increased fatty acid (FA) uptake (186%; P<0.01), adiponectin and monocyte chemotactic protein-1 secretion (+142% and +149%, respectively; P<0.05) and mRNA expression of PPARγ (+206%; P<0.01), FABP4 (+164%; P<0.001), DGAT-1 (+144%; P<0.01), adiponectin (+138%; P<0.001) and CD36 (+149%; P<0.05). In primary rat adipocytes, SEC also increased FA uptake (137%; P<0.05). Pretreatment with a SEC antagonist impaired SEC-induced FA uptake and cAMP accumulation. SEC treatment simultaneously stimulated lipolysis measured as glycerol release in 3T3-L1 adipocytes and rat adipose tissue. The present results suggest that SEC is a potent modulator of adipocyte functions, demonstrating overall a role in enhanced substrate cycling.

Abstract 447: Foam Cell Formation and Triglyceride Production After Incubation of Human Monocytes and Endothelial Cells with MAA-Modified Proteins

Introduction: Oxidized proteins and the formation of foam cells are central aspects in the development and progression of atherosclerosis. Malondialdehyde acetaldehyde (MAA) modified low density lipoprotein (LDL) is generated when lipoproteins are exposed to the oxidative product malondialdehyde. These MAA-adducted proteins have been shown to bind scavenger receptors, up-regulate adhesion molecules, induce cytokine expression, and initiate antibody and T-cell responses in both human and animal models of atherosclerotic disease. However, the induction of foam cells using these antigens has been untested. Objective: The purpose of this study was to evaluate the ability of MAA-adducted proteins on LDL to induce foam cell formation and upregulate innate inflammatory mediators of atherosclerosis in human peripheral monocytes, mouse macrophage, and human aortic endothelial cell lines. Methods: Mouse aortic endothelial (2167), mouse macrophage (J774) and human peripheral monocytes were incubated with LDL, oxidized-LDL, MAA-LDL, or MAA-human albumin (25 and 50 ng/ml) for 24 or 48 hours and evaluated. Cells were accessed for uptake using Oil Red O, triglyceride production, and cholesterol esters using filipin. Real-time semi-quantitative RT-PCR and ELISA was performed to evaluate IL-6 and monocyte chemotactic protein-1 (MCP-1) mRNA expression and protein secretion. MAA binding was also assessed using an anti-MAA monoclonal antibody. Results: LDL uptake is increased in aortic endothelial cells, J774 and human monocytes as evidenced by filipin and Oil Red O staining for esterified cholesterol LDL as compared to sham controls. Triglyceride data show a 5.1 fold increase in monocytes and a 6 fold increase in aortic endothelial cells increase following MAA-LDL incubation over control p&lt;0.001. Additionally, IHC for MAA-adducted proteins after incubation with MAA-modified demonstrate surface binding and intracellular uptake of MAA-adducted proteins. Incubation of MAA-albumin and MAA-LDL with aortic endothelial cells results in significant IL-6 and MCP-1 mRNA synthesis and protein expression 5 fold for IL-6 p&lt;0.001 and 3 fold for MCP-1 p&lt;0.01 Conclusions: These data show that MAA-modified proteins bind to and are processed by endothelial cells, macrophage, and human monocytes. MAA-modified proteins result in the production of innate immune mediators of tissue inflammation and inflammation recruitment. The direct effect of MAA in forming “foam cells” as evidenced by increased triglyceride and cholesterol is supportive for a role of MAA-modified proteins in the progression of atherosclerosis. The nature and impact of MAA-modification is hypothesized to be central to cellular apoptosis, autophagy and/or necrosis. Implications: MAA-modified proteins may be directly pathogenic in the development of plaque inflammation and progression of atherosclerosis.

Disruption of Smad4 impairs TGF-β/Smad3 and Smad7 transcriptional regulation during renal inflammation and fibrosis in vivo and in vitro

The mechanism by which TGF-β regulates renal inflammation and fibrosis is largely unclear; however, it is well accepted that its biological effects are mediated through Smad2 and Smad3 phosphorylation. Following activation, these Smads form heteromeric complex with Smad4 and translocate into the nucleus to bind and regulate the expression of target genes. Here we studied the roles of Smad4 to regulate TGF-β signaling in a mouse model of unilateral ureteral obstruction using conditional Smad4 knockout mice and in isolated Smad4 mutant macrophages and fibroblasts. Disruption of Smad4 significantly enhanced renal inflammation as evidenced by a greater CD45(+) leukocyte and F4/80(+) macrophage infiltration and upregulation of IL-1β, TNF-α, MCP-1, and ICAM-1 in the obstructed kidney and in IL-1β-stimulated macrophages. In contrast, deletion of Smad4 inhibited renal fibrosis and TGF-β1-induced collagen I expression by fibroblasts. Further studies showed that the loss of Smad4 repressed Smad7 transcription, leading to a loss of functional protein. This, in turn, inhibited IκBα expression but enhanced NF-κB activation, thereby promoting renal inflammation. Interestingly, deletion of Smad4 influenced Smad3-mediated promoter activities and the binding of Smad3 to the COL1A2 promoter, but not Smad3 phosphorylation and nuclear translocation, thereby inhibiting the fibrotic response. Thus, Smad4 may be a key regulator for the diverse roles of TGF-β1 in inflammation and fibrogenesis by interacting with Smad7 and Smad3 to influence their transcriptional activities in renal inflammation and fibrosis.

Ciclesonide inhibits TNFα- and IL-1β-induced monocyte chemotactic protein-1 (MCP-1/CCL2) secretion from human airway smooth muscle cells

Monocyte chemotactic protein-1 (MCP-1) is a member of the CC family of cytokines. It has monocyte and lymphocyte chemotactic activity and stimulates histamine release from basophils. MCP-1 is implicated in the pathogenesis of inflammatory diseases, including asthma. The airway smooth muscle (ASM) layer is thickened in asthma, and the growth factors and cytokines secreted by ASM cells play a role in the inflammatory response of the bronchial wall. Glucocorticoids and β2-agonists are first-line drug treatments for asthma. Little is known about the effect of asthma treatments on MCP-1 production from human ASM cells. Here, we determined the effect of ciclesonide (a glucocorticoid) and formoterol (a β2-agonist) on MCP-1 production from human ASM cells. TNFα and IL-1β induced MCP-1 secretion from human ASM cells. Formoterol had no effect on MCP-1 expression, while ciclesonide significantly inhibited IL-1β- and TNFα-induced MCP-1. Furthermore, ciclesonide inhibited IL-1β- and TNFα-induced MCP-1 mRNA and IL-1β- and TNFα-induced MCP-1 promoter and enhancer luciferase reporters. Western blots showed that ciclesonide had no effect on IκB degradation. Finally, ciclesonide inhibited an NF-κB luciferase reporter. Our data show that ciclesonide inhibits IL-1β- and TNFα-induced MCP-1 production from human ASM cells via a transcriptional mechanism involving inhibition of NF-κB binding.

Gsdma3 Mutation Causes Bulge Stem Cell Depletion and Alopecia Mediated by Skin Inflammation

Primary cicatricial alopecias (PCAs) are a group of permanent hair loss disorders, of which the pathogenesis is still poorly understood. The alopecia and excoriation (AE) mouse strain is a dominant mutant generated from ethyl nitrosourea mutagenesis. AE mice exhibit a progressive alopecia phenotype similar to that seen in PCAs, resulting from a point mutation in the gasdermin A3 gene. Mutant mice begin to show alopecia on the head from postnatal day 22 and experience complete hair loss by the age of 6 months, along with hyperkeratosis and catagen delay. The results of a histological examination showed that bulge stem cells in AE skin are gradually depleted, as indicated by decreased keratin 15 and CD34 expression, and reduced bromodeoxyuridine label-retaining cells in the AE bulge. In addition, AE mice display an inflammatory condition in the skin from postnatal day 7, including elevated tumor necrosis factor-α and monocyte chemotactic protein-1 mRNA levels and significantly increased macrophages and dendritic cell number. Immune privilege in the bulge was also compromised in AE skin. Consistently, after treatment with the immunosuppressive agent, cyclosporine A, immune privilege collapse, stem cell destruction, and alopecia phenotype of AE mice were all rescued. Collectively, our data demonstrate that immune-mediated destruction of bulge stem cells plays a crucial role in the pathogenesis of alopecia in AE mice, and this strain might be an interesting model for PCAs, especially for lichen planopilaris.

Age-related expression of MCP-1 and MMP-3 in mouse intervertebral disc in relation to TWEAK and TNF-α stimulation

This study was undertaken to investigate the age-related differences of monocyte chemotactic protein-1 (MCP-1) and matrix metalloproteinase-3 (MMP-3) expression in mouse intervertebral disc (IVD) and to determine whether MMP-3 plays a role in disc degeneration. Expression of MCP-1 and MMP-3 mRNA in mouse IVD was assessed by quantitative PCR. The ability of MCP-1 and MMP-3 expression in IVD to respond to TNF-α or TWEAK stimulation was examined by quantitative PCR, WB, ELISA, and immunohistochemistry. IVD derived from MMP-3-deficient and wild-type mice were compared using Safranin-O staining and immunohistochemistry. mRNA levels of MCP-1 and MMP-3 in IVD significantly diminished and the ability of MCP-1 or MMP-3 expression to respond to TNF-α or TWEAK stimulation was significantly reduced as age increased. IVD derived from 64-week-old wild-type mice showed clearly diffuse proteoglycan loss by Safranin-O staining and immunohistochemistry compared with younger mice. However, no loss of proteoglycan and typeII collagen were observed in IVD derived from 64-week-old MMP-3-deficient mice. MCP-1 and MMP-3 expression in mouse IVD showed age-related decreases. The response to inflammation in IVD also displayed age-related changes. Therefore, disc degeneration may vary with the patients' age and targeting MMP-3 may be a possible future therapeutic strategy for disc degeneration.