Wesley M. Souza

Ação da uleína sobre a produção de óxido nítrico em células RAEC e B16F10

The influence of the rich alkaloidal fraction and of the major substance in this fraction, uleine, isolated from the barks of Himatanthus lancifolius (Muell. Arg.) Woodson, Apocynaceae, popularly known as agoniada, on the nitric oxide production in RAEC and B16F10 cells and its correlation with the antioxidant activity, were investigated. For the antioxidant activity the methods of formation of a phosphomolybdenum complex and the reduction of the free radical DPPH were used. The results demonstrated an antioxidant activity of 59.3 ± 0.8% for the alkaloidal fraction, while for uleine the effect was of 0.5 ± 0.1% in the reduction of the phosphomolibdenium method. In the assay of DPPH, the alkaloidal fraction presented IC50 = 196.3 ± 8.9 µg/mL and for uleine, 6475.0 ± 25.0 µg/mL. Uleine also stimulated a maximum nitric oxide production in the concentrations of 0.1 µg/mL (20.9 ± 1.4 µM) and 1 µg/mL (41.1 ± 0.2 µM) using RAEC and B16F10 cells, respectively, demonstrating that the effect of uleine in the cells occurs by promoting the nitric oxide production pathway, but not through a free radical scavenger effect.

Effects of Himatanthus lancifolius on human leukocyte chemotaxis and their adhesion to integrins.

The aim of this work was to investigate the anti-inflammatory activities of the uleine-rich fraction extracted from the barks of Himatanthus lancifolius (Muell. Arg.) Woodson (Apocynaceae). To achieve this, we focused on its in vitro effects on some steps of the inflammatory response using peripheral human leukocytes. The results presented herein show that the uleine-rich fraction significantly inhibits the migration of casein-induced granulocytes and their adhesion to fibronectin and vitronectin, along with mononuclear cells, by down-regulating the expression of alpha 4beta1 and alpha5beta1 integrins. The data suggest that H. LANCIFOLIUS has the potential of interferring with leukocyte trafficking through its uleine-rich fraction, emphasizing its usefulness in inflammatory conditions. DEXA:dexamethasone disodium phosphate FN:fibronectin PMN:polymorphonuclear URF:uleine-rich fraction VN:vitronectin.

p-cresol mas não p-cresil sulfato estimulam a produção de MCP-1 via NF-κB p65 em células vasculares musculares lisas humanas

INTRODUCTION p-cresol (PC) and p-cresyl sulfate (PCS) are responsible for many of the uremia clinical consequences, such as atherosclerosis in Chronic Kidney Disease (CKD) patients. OBJECTIVES We investigate the in vitro impact of PC and PCS on monocyte chemoattractant protein-1 (MCP-1) expression via NF-kappa B (NF-κB) p65 in VSMC. METHODS PCS was synthesized by PC sulfatation. VSMC were extracted by enzymatic digestion of umbilical cord vein and characterized by immunofluorescence against α-actin antibody. The cells were treated with PC and PCS at their normal (n), uremic (u) and maximum uremic concentrations (m). Cell viability was assessed by MTT. MCP-1 expression was investigated by ELISA in cells supernatants after toxins treatment with or without the NF-κB p65 inhibitor. RESULTS There was no significant difference in cell viability after toxins treatment for all concentrations tested. There was a significant increase in MCP-1 expression in cells treated with PCu and PCm (p < 0.001) and PCSn, PCSu and PCSm (p < 0.001), compared with the control. When VSMC were treated with the NF-κB p65 inhibitor plus PCu and PCm, there was a significant decrease in MCP-1 production (p < 0.005). This effect was not observed with PCS. CONCLUSIONS VSMC are involved in atherosclerosis lesion formation and production of MCP-1, which contributes to the inflammatory response initiation. Our results suggest that PC mediates MCP-1 production in VSMC, probably through NF-κB p65 pathway, although we hypothesize that PCS acts through a different subunit pathway since NF-κB p65 inhibitor was not able to inhibit MCP-1 production.

Effect of PKC-β Signaling Pathway on Expression of MCP-1 and VCAM-1 in Different Cell Models in Response to Advanced Glycation End Products (AGEs)

Advanced glycation end products (AGEs) are compounds classified as uremic toxins in patients with chronic kidney disease that have several pro-inflammatory effects and are implicated in the development of cardiovascular diseases. To explore the mechanisms of AGEs–endothelium interactions through the receptor for AGEs (RAGE) in the PKC-β pathway, we evaluated the production of MCP-1 and VCAM-1 in human endothelial cells (HUVECs), monocytes, and a coculture of both. AGEs were prepared by albumin glycation and characterized by absorbance and electrophoresis. The effect of AGEs on cell viability was assessed with an MTT assay. The cells were also treated with AGEs with and without a PKC-β inhibitor. MCP-1 and VCAM-1 in the cell supernatants were estimated by ELISA, and RAGE was evaluated by immunocytochemistry. AGEs exposure did not affect cell viability, but AGEs induced RAGE, MCP-1, and VCAM-1 expression in HUVECs. When HUVECs or monocytes were incubated with AGEs and a PKC-β inhibitor, MCP-1 and VCAM-1 expression significantly decreased. However, in the coculture, exposure to AGEs and a PKC-β inhibitor produced no significant effect. This study demonstrates, in vitro, the regulatory mechanisms involved in MCP-1 production in three cellular models and VCAM-1 production in HUVECs, and thus mimics the endothelial dysfunction caused by AGEs in early atherosclerosis. Such mechanisms could serve as therapeutic targets to reduce the harmful effects of AGEs in patients with chronic kidney disease.

Sevelamer reduces endothelial inflammatory response to advanced glycation end products

Abstract Background Advanced glycation end products (AGEs) have been related to the pathogenesis of cardiovascular diseases (CVD), chronic kidney disease (CKD) and diabetes mellitus. We sought to investigate the binding capacity of sevelamer to both AGEs and uremic serum in vitro and then test this pharmaceutical effect as a potential vascular anti-inflammatory strategy. Methods AGEs were prepared by albumin glycation and characterized by absorbance and electrophoresis. Human endothelial cells were incubated in culture media containing AGEs and uremic serum with or without sevelamer. Receptor for advanced glycation end product (RAGE) expression was evaluated through immunocytochemistry and western blot to explore the interactions between AGEs and the endothelium. Inflammatory and endothelial dysfunction biomarkers, such as interleukin 6 (IL-6) and IL-8, monocyte chemoattractant protein-1 (MCP-1), plasminogen activator inhibitor-1 (PAI-1) and serum amyloid A (SAA) were also measured in cell supernatant. The chemotactic property of the supernatant was evaluated. Results AGEs significantly induced the expression of RAGE, inflammatory and endothelial activation biomarkers [IL-6, (P < 0.005); IL-8, MCP-1, PAI-1 and SAA (P < 0.001)] and monocyte chemotaxis as compared with controls. In addition, AGEs increased the levels of inflammatory biomarkers, which were observed after 6 h of endothelial cell incubation with uremic serum [IL-6 (P < 0.001) IL-8, MCP-1 and PAI-1 (P < 0.05)]. On the other hand, after 6 h of endothelial cell treatment with sevelamer, RAGE expression (P < 0.05) and levels of inflammatory biomarkers [IL-6 and IL-8 (P < 0.001), MCP-1 (P < 0.01), PAI-1 and SAA (P < 0.005)] significantly decreased compared with the AGEs/uremic serum treatment alone. Conclusions Sevelamer decreased both endothelial expression of RAGE and endothelial dysfunction biomarkers, induced by AGEs, and uremic serum. Further studies are necessary for a better understanding of the potential protective role of sevelamer on uremic serum and AGEs-mediated endothelial dysfunction.

Role of Organic Anion Transporters in the Uptake of Protein-Bound Uremic Toxins by Human Endothelial Cells and Monocyte Chemoattractant Protein-1 Expression

Organic anion transporters (OATs) are involved in the uptake of uremic toxins such as p-cresyl sulfate (PCS) and indoxyl sulfate (IS), which play a role in endothelial dysfunction in patients with chronic kidney diseases (CKD). In this study, we investigated the role of OAT1 and OAT3 in the uptake of PCS and IS into human endothelial cells. PCS was synthesized via p-cresol sulfation and characterized using analytical methods. The cells were treated with PCS and IS in the absence and presence of probenecid (Pb), an OAT inhibitor. Cell viability was assessed using the MTT assay. The absorbed toxins were analyzed using chromatography, OAT expression using immunocytochemistry and western blot, and monocyte chemoattractant protein-1 (MCP-1) expression using enzyme-linked immunosorbent assay. Cell viability decreased after toxin treatment in a dose-dependent manner. PCS and IS showed significant internalization after 60 min treatment, while no internalization was observed in the presence of Pb, suggesting that OATs are involved in the transport of both toxins. Immunocytochemistry and western blot demonstrated OAT1 and OAT3 expression in endothelial cells. MCP-1 expression increased after toxins treatment but decreased after Pb treatment. PCS and IS uptake were mediated by OATs, and OAT blockage could serve as a therapeutic strategy to inhibit MCP-1 expression.