Asiatic acid stabilizes cytoskeletal proteins and prevents TNF-α-induced disorganization of cell-cell junctions in human aortic endothelial cells

Endothelial hyperpermeability represents an initiating step in early atherosclerosis and it often occurs as a result of endothelial barrier dysfunction. Asiatic acid, a major triterpene isolated from Centella asiatica (L.) Urban, has previously been demonstrated to protect against tumor necrosis factor (TNF)-α-induced endothelial barrier dysfunction. The present study aimed to investigate the mechanisms underlying the barrier protective effect of asiatic acid in human aortic endothelial cells (HAECs). The localization of F-actin, diphosphorylated myosin light chain (diphospho-MLC), adherens junctions (AJs) and tight junctions (TJs) was studied using immunocytochemistry techniques and confocal microscopy. Their total protein expressions were examined using western blot analysis. The endothelial permeability was assessed using In Vitro Vascular Permeability Assay kits. In addition, intracellular redistribution of the junctional proteins was evaluated using subcellular fractionation kits. We show that asiatic acid stabilized F-actin and diphospho-MLC at the cell periphery and prevented their rearrangement stimulated by TNF-α. However, asiatic acid failed to attenuate cytochalasin D-induced increased permeability. Besides, asiatic acid abrogated TNF-α-induced structural reorganization of vascular endothelial (VE)-cadherin and β-catenin by preserving their reticulum structures at cell-cell contact areas. In addition, asiatic acid also inhibited TNF-α-induced redistribution of occludin and zona occludens (ZO)-1 in different subcellular fractions. In conclusion, the barrier-stabilizing effect of asiatic acid might be associated with preservation of AJs and prevention of TJ redistribution caused by TNF-α. This study provides evidence to support the potential use of asiatic acid in the prevention of early atherosclerosis, which is initiated by endothelial barrier dysfunction

Asiaticoside inhibits TNF-alpha-induced endothelial hyperpermeability of human aortic endothelial cells

The increase in endothelial permeability often promotes edema formation in various pathological conditions. Tumor necrosis factor-alpha (TNF-α), a pro-atherogenic cytokine, impairs endothelial barrier function and causes endothelial dysfunction in early stage of atherosclerosis. Asiaticoside, one of the triterpenoids derived from Centella asiatica, is known to possess antiinflammatory activity. In order to examine the role of asiaticoside in preserving the endothelial barrier, we assessed its effects on endothelial hyperpermeability and disruption of actin filaments evoked by TNF-α in human aortic endothelial cells (HAEC). TNF-α caused an increase in endothelial permeability to fluorescein isothiocyanate (FITC)-dextran. Asiaticoside pretreatment significantly suppressed TNF-α-induced increased permeability. Asiaticoside also prevented TNF-α-induced actin redistribution by suppressing stress fiber formation. However, the increased F to G actin ratio stimulated by TNF-α was not changed by asiaticoside. Cytochalasin D, an actin depolymerizing agent, was used to correlate the anti-hyperpermeability effect of asiaticoside with actin cytoskeleton. Surprisingly, asiaticoside failed to prevent cytochalasin D-induced increased permeability. These results suggest that asiaticoside protects against the disruption of endothelial barrier and actin rearrangement triggered by TNF-α without a significant change in total actin pool. However, asiaticoside seems to work by other mechanisms to maintain the integrity of endothelial barrier rather than stabilizing the F-actin organization

Cryptotanshinone inhibits TNF-α-induced early atherogenic events in vitro

Endothelial dysfunction has been implicated in the pathogenesis of atherosclerosis. Salvia miltiorrhiza (danshen) is a traditional Chinese medicine that has been effectively used to treat cardiovascular disease. Cryptotanshinone (CTS), a major lipophilic compound isolated from S. miltiorrhiza, has been reported to possess cardioprotective effects. However, the anti-atherogenic effects of CTS, particularly on tumor necrosis factor-α (TNF-α)-induced endothelial cell activation, are still unclear. This study aimed to determine the effect of CTS on TNF-α-induced increased endothelial permeability, monocyte adhesion, soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), monocyte chemoattractant protein 1 (MCP-1) and impaired nitric oxide production in human umbilical vein endothelial cells (HUVECs), all of which are early events occurring in atherogenesis. We showed that CTS significantly suppressed TNF-α-induced increased endothelial permeability, monocyte adhesion, sICAM-1, sVCAM-1 and MCP-1, and restored nitric oxide production. These observations suggest that CTS possesses anti-inflammatory properties and could be a promising treatment for the prevention of cytokine-induced early atherogenesis

Barrier protective effect of asiatic acid in TNF-α-induced activation of human aortic endothelial cells

Background: Endothelial cell activation is characterized by increased endothelial permeability and increased expression of cell adhesion molecules (CAMs). This allows monocyte adherence and migration across the endothelium to occur and thereby initiates atherogenesis process. Asiatic acid is a major triterpene isolated from Centella asiatica (L.) Urban and has been shown to possess anti-oxidant, anti-hyperlipidemia and anti-inflammatory activities. Purpose: We aimed to investigate protective effects of asiatic acid on tumor necrosis factor-α (TNF-α)-induced endothelial cell activation using human aortic endothelial cells (HAECs). Study design: For cell viability assays, HAECs were treated with asiatic acid for 24 h. For other assays, HAECs were pretreated with various doses of asiatic acid (10–40 µM) for 6 h followed by stimulation with TNF-α (10 ng/ml) for 6 h. Methods: Fluorescein isothiocyanate (FITC)-dextran permeability assay was performed using commercial kits. Total protein expression of CAMs such as E-selectin, ICAM-1, VCAM-1 and PECAM-1 as well as phosphorylation of IκB-α were determined using western blot. The levels of soluble form of CAMs were measured using flow cytometry. Besides, we also examined the effects of asiatic acid on U937 monocyte adhesion and monocyte migration in HAECs using fluorescent-based assays. Results: Asiatic acid significantly suppressed endothelial hyperpermeability, increased VCAM-1 expression and increased levels of soluble CAMs (sE-selectin, sICAM-1, sVCAM-1 and sPECAM-1) triggered by TNF-α. Neither TNF-α nor asiatic acid affects PECAM-1 expression. However, asiatic acid did not inhibit TNF-α-induced increased monocyte adhesion and migration. Interestingly, asiatic acid suppressed increased phosphorylation of IκB-α stimulated by TNF-α. Conclusion: These results suggest that asiatic acid protects against endothelial barrier disruption and this might be associated with the inhibition of NF-κB activation. We have demonstrated a novel protective role of asiatic acid on endothelial function. This reveals the possibility to further explore beneficial effects of asiatic acid on chronic inflammatory diseases that are initiated by endothelial cell activation

Experimental review on the Substance P-enhanced endothelial permeability in human umbilical vein endothelial cells (HUVECS)

Inflammation is the immediate response to tissue damage or harmful stimuli. Though inconvenient, its role is significant and important as the protective and physiological response of our body. It directly sets the stage for tissue repair particularly increasing endothelial permeability which then contributes to the healing process. However, in some cases inflammation may progress out of control causing various inflammatory diseases. Neurogenic inflammation is a sub-set of inflammation and is characterized by an increase in neuronal chemical mediators such as Substance P (SP). In this study, we investigated the involvement of SP in enhancing endothelial permeability on HUVECs monolayer. Neurogenic inflammation was induced through the administration of SP (1 nM to 100 nM) on HUVECs monolayer inserts, and incubated with varying short (10, 20 and 30 minutes) and longer (6, 12 and 24 hours) time-points. FITC-Dextran were finally added to cell culture inserts for 5 minutes to let the fluorescence molecule pass through the gaps. Endothelial permeability is directly proportional with extravasation of FITC-Dextran, determined by fluorescence intensity reading. Based on our data, there were no significant differences between control group (non-treated cell) and the different concentrations of SP at different time-points. Our current findings suggest that SP was unable to increase the endothelial permeability on HUVECs monolayer inflammatory experimental model. Experiments that use this model to mimic vascular inflammation in laboratory settings may require further elucidation in the future

Effect of plasma treatment (He/CH4) on the glass surface for the reduction of powder flux adhesion in the spray drying process

A 50Hz glow discharge He/CH4 plasma was generated and applied for the glass surface modification to reduce the powder adhesion on wall of spray dryer. The hydrophobicity of the samples determined by the water droplet contact angle and adhesion weight on glass, dependent on the CH4 flow rate and plasma exposure time. The presence of CH3 groups and higher surface roughness of the plasma treated glass were the factors for its hydrophobicity development. Response surface methodology (RSM) results using central composite rotatable design (CCRD) showed that optimal responses were obtained by the combination of parameters, CH4 gas flow rate = 3 sccm and exposure time = 10 min. In optimum conditions, the contact angle increased by 47% and the weight of the adhesion reduced by 38% (w/w). The plasma treatment could enhance the value of the contact angle and thus reduced the adhesion on the spray dryer glass surface

Barrier protective effects of asiatic acid in TNF-α-induced activation of human aortic endothelial cells

Background: Endothelial cell activation is characterized by increased endothelial permeability and increased expression of cell adhesion molecules (CAMs). This allows monocyte adherence and migration across the endothelium to occur and thereby initiates atherogenesis process. Asiatic acid is a major triterpene isolated from Centella asiatica (L.) Urban and has been shown to possess anti-oxidant, anti-hyperlipidemia and anti-inflammatory activities. Purpose: We aimed to investigate protective effects of asiatic acid on tumor necrosis factor-α (TNF-α)-induced endothelial cell activation using human aortic endothelial cells (HAECs). Study design: For cell viability assays, HAECs were treated with asiatic acid for 24 h. For other assays, HAECs were pretreated with various doses of asiatic acid (10–40 µM) for 6 h followed by stimulation with TNF-α (10 ng/ml) for 6 h. Methods: Fluorescein isothiocyanate (FITC)-dextran permeability assay was performed using commercial kits. Total protein expression of CAMs such as E-selectin, ICAM-1, VCAM-1 and PECAM-1 as well as phosphorylation of IκB-α were determined using western blot. The levels of soluble form of CAMs were measured using flow cytometry. Besides, we also examined the effects of asiatic acid on U937 monocyte adhesion and monocyte migration in HAECs using fluorescent-based assays. Results: Asiatic acid significantly suppressed endothelial hyperpermeability, increased VCAM-1 expression and increased levels of soluble CAMs (sE-selectin, sICAM-1, sVCAM-1 and sPECAM-1) triggered by TNF-α. Neither TNF-α nor asiatic acid affects PECAM-1 expression. However, asiatic acid did not inhibit TNF-α-induced increased monocyte adhesion and migration. Interestingly, asiatic acid suppressed increased phosphorylation of IκB-α stimulated by TNF-α. Conclusion: These results suggest that asiatic acid protects against endothelial barrier disruption and this might be associated with the inhibition of NF-κB activation. We have demonstrated a novel protective role of asiatic acid on endothelial function. This reveals the possibility to further explore beneficial effects of asiatic acid on chronic inflammatory diseases that are initiated by endothelial cell activation

A low power 50 Hz argon plasma for surface modification of polytetrafluoroethylene

The characteristics of a low power 50 Hz argon plasma for surface treatment of polytetrafluoroethylene (PTFE) film is presented in this article. The current–voltage behavior of the discharge and time-varying intensity of the discharge showed that a DC glow discharge was generated in reversed polarity at every half-cycle. At discharge power between 0.5 and 1 W, the measured electron temperature and density were 2–3 eV and ∼108 cm−3, respectively. The optical emission spectrum of the argon plasma showed presence of some 'impurity species' such as OH, N2 and H, which presumably originated from the residual air in the discharge chamber. On exposure of PTFE films to the argon glow plasma at pressure 120 Pa and discharge power 0.5 to 1 W, the water contact angle reduced by 4% to 20% from the original 114° at pristine condition, which confirms improvement of its surface wettability. The increase in wettability was attributed to incorporation of oxygen-containing functional groups on the treated surface and concomitant reduction in fluorine as revealed by the XPS analysis and increase in surface roughness analyzed from the atomic force micrographs. Ageing upon storage in ambient air showed retention of the induced increase in surface wettability