VCAM-1 Gene Expression Research Articles

Blocking Complement Factor B Activation Reduces Renal Injury and Inflammation in a Rat Brain Death Model.

Introduction: The majority of kidneys used for transplantation are retrieved from brain-dead organ donors. In brain death, the irreversible loss of brain functions results in hemodynamic instability, hormonal changes and immunological activation. Recently, brain death has been shown to cause activation of the complement system, which is adversely associated with renal allograft outcome in recipients. Modulation of the complement system in the brain-dead donor might be a promising strategy to improve organ quality before transplantation. This study investigated the effect of an inhibitory antibody against complement factor B on brain death-induced renal inflammation and injury.Method: Brain death was induced in male Fischer rats by inflating a balloon catheter in the epidural space. Anti-factor B (anti-FB) or saline was administered intravenously 20 min before the induction of brain death (n = 8/group). Sham-operated rats served as controls (n = 4). After 4 h of brain death, renal function, renal injury, and inflammation were assessed.Results: Pretreatment with anti-FB resulted in significantly less systemic and local complement activation than in saline-treated rats after brain death. Moreover, anti-FB treatment preserved renal function, reflected by significantly reduced serum creatinine levels compared to saline-treated rats after 4 h of brain death. Furthermore, anti-FB significantly attenuated histological injury, as seen by reduced tubular injury scores, lower renal gene expression levels (>75%) and renal deposition of kidney injury marker-1. In addition, anti-FB treatment significantly prevented renal macrophage influx and reduced systemic IL-6 levels compared to saline-treated rats after brain death. Lastly, renal gene expression of IL-6, MCP-1, and VCAM-1 were significantly reduced in rats treated with anti-FB.Conclusion: This study shows that donor pretreatment with anti-FB preserved renal function, reduced renal damage and inflammation prior to transplantation. Therefore, inhibition of factor B in organ donors might be a promising strategy to reduce brain death-induced renal injury and inflammation.

Abstract 224: Sub-cytotoxic Levels of Heavy Metals Induce Pro-inflammatory Signaling in the Aortic Endothelium without Impairing Flow-Mediated Dilation in Rats

Introduction: Acute exposure to tobacco or marijuana secondhand smoke (SHS) causes endothelial dysfunction. The identity of specific SHS constituents that cause vascular toxicity is unclear. Heavy metals are present in SHS and at elevated levels in the blood of smokers, and may mediate acute endothelial dysfunction through reactive oxygen species formation and decreasing NO bioavailability. We assessed the effects of exposure to cadmium, lead, mercury, and arsenic at levels present in the blood of human smokers on flow-mediated dilation (FMD) as a measure of endothelial function in rats. We also evaluated the effects of heavy metal exposure on intimal structure, protein localization pattern, and inflammatory gene expression in the aortic endothelium. Hypothesis: Sub-cytotoxic levels of heavy metals impair FMD, alter intimal structure, and induce pro-inflammatory signaling in endothelium. Methods: We injected rats (n=8/group) with heavy metal cocktail or vehicle intravenously and quantitated pre- and post-exposure FMDs measures defined as percent vasodilation of femoral artery after transient ischemia. We performed en face aorta immunostaining and assessed endothelial cell axis alignment, cell length ratio, and localization pattern of PECAM-1, VE-cadherin, and vimentin. We also quantified gene expression of key endothelial proteins in aorta homogenates. Results: FMD was not impaired in the heavy metal group (8.8±3.6(SD)% vs. 12.9±8.0%, p=.31 or controls (7.5±2.7% vs. 8.8±5.8%, p=.63). No significant difference in cell length ratio and endothelial x and y axes of alignment were detected between groups (p>.8) and localization of PECAM-1, VE-cadherin, and vimentin in the aorta endothelium remained unaltered following heavy metal injection. However, expression of PECAM-1 and VE-cadherin was significantly lower in the heavy metal-treated rats, while VCAM-1 gene expression was significantly higher (p<.05). Conclusion: Acute exposure to sub-cytotoxic levels of heavy metals can induce pro-inflammatory signaling in endothelium, which could potentially lead to vascular injury. However, FMD and endothelial structure remain unchanged by heavy metal exposure.

MELATONIN ABOLISHES RENAL INFILTRATION OF T LYMPHOCYTES IN A HIGH SALT DIET INDUCED HYPERTENSION MODEL

Objective: Low grade inflammation and oxidative stress play a role in the pathogenesis of cardiovascular diseases, including hypertension (HTN). There is emerging evidence that the immune system, and particularly the adaptive immune response, may be involved in the triggering of these processes. In a previous study we demonstrated that melatonin prevents kidney damage in salt induced HTN model by decreasing oxidative stress. We hypothesized that this effect is involving the immunomodulation properties of melatonin. Design and method: Dahl salt sensitive (DSS) rats were fed normal chow [control (Ctrl)], or high salt diet (HSD) or HSD and melatonin (Mel) (30/mg/kg/day) in their water for 8 weeks (n = 8 each group). By the end of the treatment or when rats were considered ill with less than 24 hours survival, rats were sacrificed and kidneys were harvested for immediate lymphocytes isolation and characterization by FACS (CD3+CD4+ and CD3+CD8+) and for chemoatractants (vcam-1, cxcl10,cxcl1,cx3cl1,cxcl12,ccl3 and nrlp3) gene expression studies. Results: HSD was associated with significant renal infiltration of CD4+ and CD8+ T lymphocytes compare to control (0.16 ± 0.02 vs. 5.18 ± 1.6 and 0.95 ± 0.15 vs. 4.6 ± 0.75, % of CD3+ cells, Ctrl vs. HSD, CD4+ and CD8+ respectively, p < 0.05). Melatonin treatment significantly reduced renal lymphocytes infiltration (0.7 ± 0.2 and 1.37 ± 0.45 % of CD3+ cells, CD4+ and CD8+ respectively, p < 0.05 vs. HSD). HSD increased significantly mRNA expression of renal vcam-1, cxcl10,cxcl1, ccl3 and nrlp3 (2 ± 0.2, 2.4 ± 0.3, 3.1 ± 0.4, 2 ± 0.3, 1.6 ± 0.1, time fold, respectively, HSD vs. Ctrl, p < 0.5). The mRNA expression of cxcl10 and cxcl1, which are lymphocytes attractants, was significantly low in the melatonin treated group despite concomitant salt consumption. Conclusions: Treating HSD fed rats with melatonin is associated with decrease in some chemoatractants expression and with significantly lower renal T lymphocytes infiltration.

Liraglutide Improves Renal Endothelial Function in Obese Zucker Rats on a High-Salt Diet.

Metabolic syndrome is a common risk factor in chronic kidney disease. We investigated whether liraglutide [(LIRA), a glucagon-like peptide-1 receptor (GLP-1R) agonist] treatment improved renal vascular function and renal remodeling in male Zucker rats on a high-salt diet (6% NaCl). Zucker lean (+/+) and obese (fa/fa) rats (8 weeks old) were treated with vehicle or LIRA (0.1 mg/kg per day) for 8 weeks on a high-salt diet. The glomerular filtration rate (GFR) was measured at 0 and 8 weeks using the fluorescein isothiocyanate/sinistrin method in conscious rats. We used X-ray microangiography to measure renal arterial vessel diameter (70-350 µm) and vessel number in vivo in anesthetized rats. Renal protein expression levels of nitrotyrosine, CD-68, endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), transforming growth factor-β1, cyclooxygenase-2, and GLP-1R were assessed by western blotting. Renal gene expressions were determined by real-time polymerase chain reaction. In contrast to vehicle-treated rats, fa/fa-LIRA rats improved GFR, nitric oxide (NO)-mediated vasodilation in response to acetylcholine and sodium nitroprusside in small arterial vessels (<200 µm diameter). LIRA treatment increased vessel responsivity to NO donors in comparison with vehicle treatment. Increases in the expressions of proinflammatory, profibrotic, and oxidative stress related genes in fa/fa rats relative to +/+ were unaltered by LIRA, other than a trend toward attenuation of VCAM-1 gene expression. However, LIRA treatment increased protein expressions of eNOS (P = 0.014) and VEGF (P = 0.063), while reducing glomerular macrophage infiltration in comparison with vehicle-treated fa/fa rats. Low-dose LIRA treatment improved renal vascular function through amelioration of vascular dysfunction and improved NO-mediated dilation of small intrarenal arteries and arterioles and a reduction in renal inflammation.

Rooibos (Aspalathus linearis) and honeybush (Cyclopia species) modulate the oxidative stress associated injury of diesel exhaust particles in human umbilical vein endothelial cells

BackgroundPrevious evidence show foods and beverages rich in polyphenolic compounds to have favourable effects on the cardiovascular system. HypothesisThe current study assessed the modulation of oxidative stress and associated inflammation induced by diesel exhaust particles (DEP - SRM 2975) by pre-treatment of human umbilical vein endothelial cells (HUVECs) with aqueous extracts of rooibos [fermented (FR) as well as green form (GR)] and honeybush [fermented form (FH)]. Study designHUVEC are either exposed to DEP (10 µg/ml) for 4 h or pre-treated with 40 and 60 µg/ml of FR or GH or FR, or 50 µg/ml orientin (OR) for 6 h prior to DEP exposure. MethodsIn vitro antioxidant capacity of the extracts was assessed and the polyphenol contents were also assessed by HPLC. ROS, cell viability, lactate dehydrogenase leakage, lipid peroxidation, GSH:GSSG ratios, conjugated diene and protein carbonyl levels were determined as indices of oxidative stress and cytotoxicity. RT-qPCR and western blot were used to assess inflammatory cytokines and antioxidant genes expression. ResultsDEP caused a dose and time-dependent increase in ROS production, significant (p < 0.001) increase in protein carbonyl (PC) formation, thiobarbituric acid reactive substances and conjugated dienes levels (p < 0.01) and a significant reduction in glutathione (GSH) redox status. Pre-incubation with either the herbal extracts or orientin attenuated these effects. The significant increase in IL-1α, IL-6, IL-8, VCAM-1 and ATF4 gene expression caused by DEP (10 µg/ml) were also attenuated by the presence of the FR, GR and FH extracts, and OR . Pre-treatment with the rooibos extracts or flavone orientin enhanced cell viability, reduced LDH leakage, enhanced mRNA expression of NQO1 and Nrf2, but repressed CYP1B1 mRNA induced by DEP. Western blot showed both the herbal tea extracts and orientin to enhance NQO1 and γGSC protein induction by DEP. ConclusionTaken together, the herbal extracts offer protection against DEP-induced oxidative stress and inflammatory response.

Heme oxygenase-1 induction by rosiglitazone via PKCα/AMPKα/p38 MAPKα/SIRT1/PPARγ pathway suppresses lipopolysaccharide-mediated pulmonary inflammation

HO-1 (heme oxygenase-1), an antioxidant enzyme, induced by rosiglitazone (PPAR ligands) can be a potential treatment of inflammation. However, the mechanisms of rosiglitazone-induced HO-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain largely unknown. In this study, we found that upregulation of HO-1 in vitro or in vivo by rosiglitazone attenuated VCAM-1 gene expression and monocyte adhesion to HPAEpiCs challenged with lipopolysaccharide (LPS). The inhibitory effects of rosiglitazone on LPS-mediated responses were reversed by transfection with HO-1 siRNA. LPS-induced VCAM-1 expression was mediated through NF-κB activation which was attenuated by rosiglitazone via suppressing p65 activation and translocation into the nucleus. Moreover, pretreatment with the inhibitor of PKCs (H7), PKCα (Gö6976), AMPKα (Compound C), p38 MAPKα (p38i VIII), SIRT1 (Sirtinol), or PPARγ (T0070907) and transfection with siRNA of PKCα, AMPKα, p38 MAPKα, SIRT1, or PPARγ abolished the rosiglitazone-induced HO-1 expression in HPAEpiCs. Further studies indicated that rosiglitazone stimulated SIRT1 deacetylase leading to PGC1α translocation from the cytosol into the nucleus, promoting fragmentation of NCoR and phosphorylation of PPARγ. Subsequently, PPARγ was activated by phosphorylation of PKCα, AMPKα, p38 MAPKα, and SIRT1, which turned on transcription of HO-1 gene by binding to PPAR response element (PPRE) and enhancing PPARγ promoter activity. These results suggested that rosiglitazone-induced HO-1 expression is mediated through PKCα/AMPKα/p38 MAPKα/SIRT1-dependent deacetylation of Ac-PGC1α and fragmentation of NCoR/PPARγ activation in HPAEpiCs. Up-regulation of HO-1 protected against the inflammatory responses triggered by LPS, at least in part, through attenuation of NF-κB.

Angiotensin-(1-7) Inhibits Thrombin-Induced Endothelial Phenotypic Changes and Reactive Oxygen Species Production via NADPH Oxidase 5 Downregulation.

Background and Aims: The angiotensin-(1-7)/angiotensin-converting enzyme 2/Mas receptor axis counter-regulates the detrimental effects of angiotensin II. Beneficial effects of angiotensin-(1-7), including anti-inflammation, oxidative stress reduction, and anti-thrombosis, have been reported. Previous studies documented that ramipril decreased thrombin generation in human hypertension and that the anti-thrombotic effects of captopril and losartan were angiotensin-(1-7)-dependent, suggesting an interaction between thrombin and angiotensin-(1-7). However, it is not clear whether angiotensin-(1-7) can alleviate the endothelial phenotypic changes induced by thrombin. We have previously documented cytoskeleton remodeling, cell adhesion, and cell migration as dominant altered phenotypes in thrombin-stimulated human aortic endothelial cells (HAECs). In this study, we investigated whether angiotensin-(1-7) can modulate thrombin-induced phenotypic changes. Furthermore, we investigated whether NAPDH oxidase 5 (Nox5)-produced reactive oxygen species (ROS) play a significant role in angiotensin-(1-7)-mediated phenotypic changes.Methods: HAECs were pretreated with 100 nM angiotensin-(1-7) for 1 h, followed by stimulation with 2 units/mL thrombin for different times. Immunofluorescent assay, monocyte adhesion assay, wound-healing assay, ROS assay, real-time PCR, Western blotting, and Nox5 siRNA transfection were conducted. HAECs were pretreated with the ROS scavenger N-acetylcysteine (NAC) to determine whether thrombin-induced phenotypic changes depended on ROS production.Results: Angiotensin-(1-7) prevented thrombin-induced actin cytoskeleton derangements, monocyte adhesion, and migratory impairment. Nox5 siRNA transfection confirmed that thrombin-induced Nox5 expression stimulated ROS production and increased HO-1/NQO-1/ICAM-1/VCAM-1 gene expression, all of which were decreased by angiotensin-(1-7). Phenotypic changes induced by thrombin were prevented by NAC pretreatment.Conclusion: Angiotensin-(1-7) prevents actin cytoskeleton derangement, monocyte adhesion, and migration impairment induced by thrombin via downregulation of ROS production. In addition, thrombin-induced Nox5 expression is involved in the production of ROS, and angiotensin-(1-7) decreases ROS through its inhibitory effect on Nox5 expression.

Study of VCAM-1 Gene Expression in Normal and Tumoral Tissues in Patients with Colorectal Cancer

Study of VCAM-1 Gene Expression in Normal and Tumoral Tissues in Patients with Colorectal Cancer

Study of VCAM-1 Gene Expression in Normal and Tumoral Tissues in Patients with Colorectal Cancer

Study of VCAM-1 Gene Expression in Normal and Tumoral Tissues in Patients with Colorectal Cancer

Trans Fatty Acids Suppress TNF‐α‐Induced Inflammatory Gene Expression in Endothelial (HUVEC) and Hepatocellular Carcinoma (HepG2) Cells

Trans fatty acids (TFA) intake has been linked to cardiovascular diseases and liver diseases; yet the effect of TFA on inflammation remains controversial. Accordingly, the objective of this paper was to determine the in vitro effects of TFA on inflammatory gene expression. Human umbilical vein endothelial cells (HUVEC) and human hepatocellular carcinoma (HepG2) cells were treated for 24h with either trans-vaccenic acid (tVA), trans-palmitoleic acid (tPA) or elaidic acid (EA) at concentrations of 5-150µM, or with a mixture of tVA and tPA (150/50µM). All TFA were highly incorporated into cell membranes, as determined by gas chromatography, representing 15-20% of total fatty acids in HUVEC and 3-8% in HepG2 cells. Incorporation of EA, a common industrial TFA, increased the ratio of the stearoyl-CoA desaturase (SCD-1), a key enzyme involved in fatty acid metabolism. Ruminant TFA, including tVA, tPA and the mixture of tVA and tPA, significantly reduced the TNF-α-induced gene expression of TNF, VCAM-1 and SOD2 in HUVEC, as well as TNF and IL-8 in HepG2 cells. EA also decreased inflammatory gene expression in HUVEC, but not in HepG2 cells. The inhibition of peroxisome proliferator-activated receptor (PPAR)-γ did not influence the effects of TFA on gene expression. Overall, physiological and supraphysiological concentrations of TFA, especially tVA and tPA, prevented inflammatory gene expression in vitro. This effect is independent of PPAR-γ activation and may be due to an alteration of fatty acid metabolism in cell membranes caused by the high incorporation of TFA.

Klotho attenuated antibody-mediated porcine endothelial cell activation and injury.

Long-term success in pig-to-primate xenotransplantation is currently hampered by acute vascular rejection (AVR), characterized by endothelial cell (EC) activation and injury. Klotho has anti-apoptotic, anti-inflammatory effects on EC and protects EC against reactive oxygen species, rendering klotho a promising molecule to control AVR. In this study, porcine ECs were pre-incubated with klotho and then exposed to xenoreactive antibodies and complement. Real-time PCR revealed that klotho suppressed antibody-induced pro-inflammatory gene expression of VCAM-1 and IL-1α. NF-κB activation, IκBα phosphorylation, was also attenuated by klotho administration. Furthermore, klotho induced in porcine EC resistance against complement-dependent cytotoxicity. Accompanying this change, the binding of IgG and IgM xenoreactive antibodies to porcine EC was decreased and the expression of anti-inflammatory gene HO-1 was upregulated. These findings indicated that klotho protein protected porcine EC from activation and injury caused by binding of xenoreactive antibodies and was a promising candidate molecule in a multitransgenic pig strategy for xenotransplantation.

Alisol B 23-acetate protects against non-alcoholic steatohepatitis in mice via farnesoid X receptor activation.

Alisol B 23-acetate (AB23A) is a natural triterpenoid isolated from the traditional Chinese medicine rhizoma alismatis, which exhibits a number of pharmacological activities, including anti-hepatitis virus, anti-cancer and antibacterial effects. In this study we examined whether AB23A protected against non-alcoholic steatohepatitis (NASH) in mice, and the mechanisms underlying the protective effects. NASH was induced in mice fed a methionine and choline-deficient (MCD) diet for 4 weeks. The mice were simultaneously treated with AB23A (15, 30, and 60 mg·kg-1·d-1, ig) for 4 weeks. On the last day, blood samples and livers were collected. Serum liver functional enzymes, inflammatoru markers were assessed. The livers were histologically examined using H&E, Oil Red O, Masson's trichrome and Sirius Red staining. Mouse primary hepatocytes were used for in vitro experiments. The mechanisms underlying AB23A protection were analyzed using siRNA, qRT-PCR, and Western blot assays. AB23A treatment significantly and dose-dependently decreased the elevated levels of serum ALT and AST in MCD diet-fed mice. Furthermore, AB23A treatment significantly reduced hepatic triglyceride accumulation, inflammatory cell infiltration and hepatic fibrosis in the mice. AB23A-induced decreases in serum and hepatic lipids were related to decreased hepatic lipogenesis through decreasing hepatic levels of SREBP-1c, FAS, ACC1 and SCD1 and increased lipid metabolism via inducing PPARα, CPT1α, ACADS and LPL. The reduction in inflammatory cell infiltration corresponded to deceased serum levels of mKC and MCP-1 and decreased hepatic gene expression of MCP-1 and VCAM-1. The reduction in hepatic fibrosis was correlated with decreased hepatic gene expression of fibrosis markers. The protective effects of AB23A were FXR-dependent, because treatment with the FXR agonist CDCA mimicked AB23A-induced hepato-protection in the mice, whereas co-administration of FXR antagonist guggulsterone abrogated AB23A-induced hepato-protection. In mouse primary hepatocytes, FXR gene silencing abrogated AB23A-induced changes in gene expression of Apo C-II, CPT1α, ACADS and LPL. AB23A produces protective effects against NASH in mice via FXR activation.

NADPH Oxidase/ROS-Dependent VCAM-1 Induction on TNF-α-Challenged Human Cardiac Fibroblasts Enhances Monocyte Adhesion.

The inflammation-dependent adhesion molecule expressions are characterized in cardiovascular diseases and myocardial tissue infiltrations. Several pro-inflammatory cytokines are elevated in the acute myocardial injury and infarction. Tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine, is raised in the injury tissues and inflammatory regions and involved in the pathogenesis of cardiac injury, inflammation, and apoptosis. In fibroblasts, TNF-α-triggered expression of vascular cell adhesion molecule (VCAM)-1 aggravated the heart inflammation. However, the mechanisms underlying TNF-α-mediated VCAM-1 expression in cardiac fibroblasts remain unclear. Here, the primary cultured human cardiac fibroblasts (HCFs) were used to investigate the effects of TNF-α on VCAM-1 expression. The molecular evidence, including protein, mRNA, and promoter analyses, indicated that TNF-α-induced VCAM-1 gene expression is mediated through the TNFR-dependent manner. Activation of TNF-α/TNFR system triggered PKCα-dependent NADPH oxidase (Nox)/reactive oxygen species (ROS) signal linking to MAPK cascades, and then led to activation of the transcription factor, AP-1. Moreover, the results of mRNA and promoter assay demonstrated that c-Jun/AP-1 phosphorylated by TNF-α turns on VCAM-1 gene expression. Subsequently, up-regulated VCAM-1 on the cell surface of TNF-α-challenged HCFs increased the number of monocytes adhering to these cells. These results indicated that in HCFs, activation of AP-1 by PKCα-dependent Nox/ROS/MAPKs cascades is required for TNF-α-induced VCAM-1 expression. To clarify the mechanisms of TNF-α-induced VCAM-1 expression in HCFs may provide therapeutic strategies for heart injury and inflammatory diseases.

Abstract 11890: S100A8/A9 is Increased in Psoriasis Serum, Relates to Vascular Diseases and Activates Human Endothelial Cells

Introduction: Psoriasis is a chronic inflammatory disorder of the skin affecting 2-4% of the population and is associated with an increased risk of cardiovascular events, specifically myocardial infarction. Methods: In a large, ongoing prospective cohort study of psoriasis and cardiovascular diseases (NCT01778569), we studied a consecutive sample (n=100) and aimed to investigate the potential role that S100A8/A9 may have in linking psoriasis to CV disease measured by FDG PET CT and coronary CTA. Furthermore, we conducted in vitro experiments on human aortic endothelial cells (ECs) to examine whether treatement with S100A9 activates these cells. We hypothesized that S100A8/A9 would relate to psoriasis severity, relate to in vivo vascular inflammation by FDG PET CT, non-calcified burden of coronary disease by CCTA and increase endothelial cell activation. Results: We observed that the S100A8/A9 heterodimer was elevated in serum (mean psoriasis: 2019 ± 100.1; non-psoriasis: 1634 ± 160.7; p = 0.02), correlating both with psoriasis severity score (adjusted β = 0.53, p = 0.02) and overall aortic vascular inflammation measured by FDG PET CT (adjusted β = 0.48, p = 0.02) beyond the Framingham Risk Score. Additionally, we found that S100A8/A9 was associated with direct coronary atherosclerosis measured by coronary CTA demonstrating an increase in total (β = 0.16, p = 0.04) and non-calcified plaque burden (β = 0.23, p = 0.003) but not dense calcified burden. When EC's were treated with S100A9, ICAM1, E-Selectin and VCAM1 gene expression levels increased 10-fold (p = 0.001), 86-fold (P = 0.007) and 20-fold (p = 0.0002) respectively compared to the untreated human aortic endothelial cells. Conclusions: S100A8/A9 related to psoriasis severity, in vivo vascular inflammation, non-calcified plaque in the coronary arteries and EC activation. These findings suggest this protein may play a role in linking psoriasis to CVD and a role in early atherosclerotic plaque formation. Ongoing studies aim to elucidate the source of S100A8/A9 in the blood and to characterize the signaling pathway utilized by S100A8/A9 to understand whether this pathway is broadly applicable to vascular diseases associated with chronic inflammation.

In vitro effect of glucocorticoids on nasal polyps

AbstractGlucocorticoids are considered the main treatment option for nasal polyps, but their effect is only recently being understood. AimTo evaluate whether fluticasone propionate (FP) inhibits the inflammatory process induced by TNF-alpha in vitro, and to assess if NF-kappaB is associated to this inhibition. Study DesignExperimental in vitro study. Materials and MethodsNasal polyp fibroblasts were cultured during 24 hours. Three different concentrations of FP (1, 10 and 100 nM, added to TNF-alpha) were compared to negative (without additive) and positive (TNF-alpha) controls. Gene expression (RTQ-PCR) and protein concentration (ELISA) of VCAM-1, ICAM-1, eotaxin and RANTES were measured, as well as the nuclear translocation of NF-kappaB. ResultsTNF-alpha significantly increased protein concentration and RNA expression of all the studied molecules, as well as the nuclear translocation of NF-kappaB, when compared to the negative control. FP decreased these parameters in a dose-dependent manner, statistically different from positive control up to 100nM. ConclusionsFP extensively inhibited inflammatory recruiters, at both protein and RNA levels, confirming the ability of glucocorticoids to modulate the inflammatory process in nasal polyps. This inhibition was associated to decreased NF-kappaB nuclear translocation, demonstrating that this is an important mechanism of glucocorticoids action for nasal polyps.

The development of a reactive brain endothelium after psychosocial stress

Psychosocial stress is associated with increased inflammation and higher prevalence of mental health disorders like anxiety and depression. Through the activation of several neuroendocrine pathways, psychological stress leads to significant physiological, immunological, and behavioral changes. Repeated social defeat (RSD), a murine model of psychosocial stress, recapitulates many of the behavioral and immunological effects observed in humans, including increased circulating cytokines, immune cell recruitment, and prolonged anxiety-like behavior. The aim of this study was to elucidate the mechanisms underlying stress-induced immune cell trafficking to the brain that leads to the development of a reactive endothelium and behavioral changes. We show that RSD caused an exposure-dependent increase in the gene expression of ICAM1, VCAM1, E-selectin, CXCL1, and CXCL2 in the brain that increased with additional days of stress. RSD-induced ICAM1 and VCAM1 protein expression were localized to the vasculature of brain regions implicated in fear and anxiety responses, which spatially corresponded to previously reported patterns of myeloid cell trafficking. Comparison between enriched CD11b+ cells (microglia/macrophages) and enriched GLAST-1+/CD11b- cells (astrocytes) revealed that RSD increased the gene expression of IL-1 beta, CCL2, and CXCL2 in microglia/macrophages, but not astrocytes. Collectively, these data indicate that critical adhesion mediators are increased in the brain vasculature following RSD. This study begins to establish a mechanism by which the brain facilitates stress-induced immune cell recruitment that may underlie anxiety and mood disorders.

Human bone marrow mesenchymal stromal cells exhibit suppressive effects on lymphocytes derived from HTLV-1 infected individuals

Human multipotent mesenchymal stromal cells (MSC) display immunoregulatory functions that can modulate innate and adaptive cellular immune responses. The suppressive and immunomodulatory activities of MSC occur by the action of soluble factors that are constitutively produced and released by these cells or alternatively, after induction of MSC stimuli in inflammatory microenvironments. During the infection by the human T cell lymphotropic virus-type 1 (HTLV-1) around 1-5% of the infected individuals will develop a chronic inflammatory disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). However, up to date it is unknown the contribution of the MSC in the inflammatory microenvironment resulting from this chronic disease. In this study, we evaluated the MSC immunosuppressive effect on HTLV-1 infected T lymphocytes. Assays using co-culture of MSC and HTLV-1+ T lymphocyte lineages resulted in a decrease of both tax gene expression and HTLV-1 p19 antigen. The reduction of the tax gene expression and the HTLV-1 p19 were at the same time associated with 1.7 fold increase of IL-6 secretion and a higher PGE2, IDO and VCAM-1 gene expression (p≤0.05). To evaluate if MSC immunoregulation can influence the proliferation of HTLV-1 infected T lymphocytes, we compared the proliferation of lymphocytes obtained from HTLV-1+ and healthy individuals co-cultured in the presence of MSC. It was observed that the lymphoproliferative inhibition by MSC on infected lymphocytes was similar compared to the cells obtained from healthy individuals. Additionally, this suppressive effect was related to a significant increase of IDO and PGE2 gene expression (p≤0.05). Furthermore, the HTLV-1 pol gene and p19 protein were less expressed after co-culturing with MSC, suggesting that the MSC immunoregulation is effective on HTLV-1 infected T cells. In conclusion, this study suggests that MSC could be involved in the immunomodulation of the HTLV-1 infected T lymphocytes. Financial Support: FAPESP, CNPq and FUNDHERP. Keywords: MSC, HTLV-1, immunoregulation, gene expression.

Anthocyanins and their physiologically relevant metabolites alter the expression of IL-6 and VCAM-1 in CD40L and oxidized LDL challenged vascular endothelial cells.

ScopeIn vitro and in vivo studies suggest that dietary anthocyanins modulate cardiovascular disease risk; however, given anthocyanins extensive metabolism, it is likely that their degradation products and conjugated metabolites are responsible for this reported bioactivity.Methods and resultsHuman vascular endothelial cells were stimulated with either oxidized LDL (oxLDL) or cluster of differentiation 40 ligand (CD40L) and cotreated with cyanidin‐3‐glucoside and 11 of its recently identified metabolites, at 0.1, 1, and 10 μM concentrations. Protein and gene expression of IL‐6 and VCAM‐1 was quantified by ELISA and RT‐qPCR. In oxLDL‐stimulated cells the parent anthocyanin had no effect on IL‐6 production, whereas numerous anthocyanin metabolites significantly reduced IL‐6 protein levels; phase II conjugates of protocatechuic acid produced the greatest effects (>75% reduction, p ≤ 0.05). In CD40L‐stimulated cells the anthocyanin and its phase II metabolites reduced IL‐6 protein production, where protocatechuic acid‐4‐sulfate induced the greatest reduction (>96% reduction, p ≤ 0.03). Similarly, the anthocyanin and its metabolites reduced VCAM‐1 protein production, with ferulic acid producing the greatest effect (>65% reduction, p ≤ 0.04).ConclusionThese novel data provide evidence to suggest that anthocyanin metabolites are bioactive at physiologically relevant concentrations and have the potential to modulate cardiovascular disease progression by altering the expression of inflammatory mediators.

Anti inflammatory and anti angiogenic effect of black raspberry extract on human esophageal and intestinal microvascular endothelial cells

Polyphenolic compounds (anthocyanins, flavonoid glycosides) in berries prevent the initiation, promotion, and progression of carcinogenesis in rat's digestive tract and esophagus, in part, via anti-inflammatory pathways. Angiogenesis has been implicated in the pathogenesis of chronic inflammation and tumorigenesis. In this study, we investigated the anti-inflammatory and anti-angiogenic effects of black raspberry extract (BRE) on two organ specific primary human intestinal microvascular endothelial cells, (HIMEC) and human esophageal microvascular endothelial cells (HEMEC), isolated from surgically resected human intestinal and donor discarded esophagus, respectively.HEMEC and HIMEC were stimulated with TNF-α/IL-1β with or without BRE. The anti-inflammatory effects of BRE were assessed based upon COX-2, ICAM-1 and VCAM-1 gene and protein expression, PGE2 production, NFκB p65 subunit nuclear translocation as well as endothelial cell–leukocyte adhesion. The anti-angiogenic effects of BRE were assessed on cell migration, proliferation and tube formation following VEGF stimulation as well as on activation of Akt, MAPK and JNK signaling pathways.BRE inhibited TNF-α/IL-1β-induced NFκB p65 nuclear translocation, PGE2 production, up-regulation of COX-2, ICAM-1 and VCAM-1 gene and protein expression and leukocyte binding in HEMEC but not in HIMEC. BRE attenuated VEGF-induced cell migration, proliferation and tube formation in both HEMEC and HIMEC. The anti-angiogenic effect of BRE is mediated by inhibition of Akt, MAPK and JNK phosphorylations.BRE exerted differential anti-inflammatory effects between HEMEC and HIMEC following TNF-α/IL-1β activation whereas demonstrated similar anti-angiogenic effects following VEGF stimulation in both cell lines. These findings may provide more insight into the anti-tumorigenic capacities of BRE in human disease and cancer.

Heat shock protein 90 stimulates rat mesenchymal stem cell migration via PI3K/Akt and ERK1/2 pathways.

The objective of this study was to determine the role of Hsp90α in regulating the migration of mesenchymal stem cells (MSCs) and to investigate the underlying mechanisms of this effect. MSCs migration was assessed by wound healing assay and transwell migration assay. Hsp90α expression was silenced in MSC by siRNA (sirHsp90α). The activity of secreted metalloproteases MMP-2 and MMP-9, and their expression levels in MSC were evaluated using gelatin zymography, Western blot analysis and real-time PCR. Gene expression of VCAM-1 and CXCR4 cytokines was evaluated by real-time PCR. Akt and ERK activity were analyzed by Western blotting using antibodies against phosphorylated forms of these proteins. Treatment with Hsp90α significantly enhanced MSC migration, and this effect was blocked by transfecting MSC with sirHsp90α. Treating the cells with recombinant human Hsp90α (rhHsp90α) enhanced gene expression and protein levels of MMP-2 and MMP-9, as well as their secretion and activity. MSC incubated with rhHsp90α exhibited increased gene expression of CXCR4 and VCAM-1. Finally, the levels of phosphorylated Akt and Erk were markedly increased by rhHsp90α treatment. These findings indicate that Hsp90α promotes MSCs migration via PI3K/Akt and ERK signaling pathways, and that this effect is possibly mediated by MMPs, SDF-1/CXCR4 pathway, and VCAM-1.