Inflammatory Responses In Human Umbilical Vein Endothelial Cells Research Articles

HMGB1 Induced Oxidative Stress and Inflammation in Endothelial Cells Exposed to Impinging Flow

Introduction: Oxidative stress and inflammation contribute to many aspects of the pathological processes involved in intracranial aneurysm (IA). However, the underlying mechanism for inducing oxidative stress and inflammation under impinging flow remains unclear. Accumulating evidence has shown that high mobility group box-1 (HMGB1) is associated with oxidative stress-related chronic diseases and inflammatory responses. Therefore, we aimed to investigate whether HMGB1 is involved in oxidative stress and inflammatory responses in endothelial cells (ECs) exposed to impinging flow. Methods: We used a modified T-chamber to simulate the in vitro situation of human umbilical vein ECs (HUVECs) subjected to impinging flow at the arterial bifurcation in order to analyze the effect of wall shear stress (WSS) on the ECs. To investigate the role of HMGB1 in this process, we transfected ECs with short hairpin RNA (shRNA) before conducting impinging flow experiments. Intracellular reactive oxygen species (ROS) were measured by flow cytometry, and malondialdehyde, glutathione, and superoxide dismutase levels were measured to assess oxidative stress. Inflammation was assessed by measuring the mRNA expression levels of IL-1β, IL-6, and IL-8 using reverse transcription-quantitative polymerase chain reaction. We also examined the cellular localization of HMGB1 by immunofluorescence. Results: Exposure of HUVECs to WSS can increase the level of oxidative stress and inflammatory response. WSS increased the expression of HMGB1 in ECs and promoted the translocation of HMGB1 from cytosol to cytoplasm. When we knocked down HMGB1, the level of oxidative stress and inflammatory response caused by WSS in ECs decreased, suggesting that HMGB1 can mediate the oxidative stress and inflammatory response in HUVECs exposed to WSS. Conclusions: HMGB1 induced oxidative stress and inflammatory response in ECs exposed to impinging flow.

The role of 14-3-3 in the progression of vascular inflammation induced by lipopolysaccharide.

To explore the role of 14-3-3 protein and the Hippo and yes-associated protein 1 (YAP) signaling pathway in lipopolysaccharide (LPS)-induced vascular inflammation. Human umbilical vein endothelial cells (HUVECs) and C57B6 mice were treated with LPS to establish cell and animal models of vascular inflammation. Lentiviral transfection, Western blot, qPCR, immunofluorescence, immunohistochemistry, co-immunoprecipitation, and enzyme-linked immunosorbent assays were used to measure inflammatory factors and expression of 14-3-3 protein and phosphorylation of YAP at S127. HUVECs and C57B6 mice were pretreated with a YAP inhibitor, Verteporfin, to observe changes in YAP expression and downstream vascular inflammation. LPS induced acute and chronic inflammatory responses in HUVECs and mice and upregulated the expression of several inflammatory factors. LPS also induced expression of 14-3-3 protein and phosphorylation of YAP at S127 in response to acute vascular inflammation and downregulated these markers in response to chronic vascular inflammation. Verteporfin reduced these LPS-induced effects on vascular inflammation. In chronic vascular inflammation, 14-3-3 protein is downregulated, which promotes inflammation by increasing Hippo/YAP nuclear translocation.

The Toll-Like Receptor 2 Ligand Pam2CSK4 Activates Platelet Nuclear Factor-κB and Bruton’s Tyrosine Kinase Signaling to Promote Platelet-Endothelial Cell Interactions

Circulating platelets establish a variety of immunological programs and orchestrate inflammatory responses at the endothelium. Platelets express the innate immunity family of Toll-like receptors (TLRs). While TLR2/TLR1 ligands are known to activate platelets, the effects of TLR2/TLR6 ligands on platelet function remain unclear. Here, we aim to determine whether the TLR2/TLR6 agonists Pam2CSK4 and FSL-1 activate human platelets. In addition, human umbilical vein endothelial cells (HUVECs) and platelets were co-cultured to analyze the role of platelet TLR2/TLR6 on inflammation and adhesion to endothelial cells. Pam2CSK4, but not FSL-1, induced platelet granule secretion and integrin αIIbβ3 activation in a concentration-dependent manner. Moreover, Pam2CSK4 promoted platelet aggregation and increased platelet adhesion to collagen-coated surfaces. Mechanistic studies with blocking antibodies and pharmacologic inhibitors demonstrated that the TLR2/Nuclear factor-κB axis, Bruton’s-tyrosine kinase, and a secondary ADP feedback loop are involved in Pam2CSK4-induced platelet functional responses. Interestingly, Pam2CSK4 showed cooperation with immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling to enhance platelet activation. Finally, the presence of platelets increased inflammatory responses in HUVECs treated with Pam2CSK4, and platelets challenged with Pam2CSK4 showed increased adhesion to HUVECs under static and physiologically relevant flow conditions. Herein, we define a functional role for platelet TLR2-mediated signaling, which may represent a druggable target to dampen excessive platelet activation in thrombo-inflammatory diseases.

Activation of κ-opioid receptor inhibits inflammatory response induced by sodium palmitate in human umbilical vein endothelial cells

ObjectivesThe current study aims to investigate the effect of κ-opioid receptor (κ-OR) activation on sodium palmitate (SP)-induced human umbilical vein endothelial cells (HUVECs) inflammatory response and elucidate the underlying mechanisms. MethodsA hyperlipidemic cell model was established and treated with κ-OR agonist (U50,488H), and antagonist (norbinaltorphimine, nor-BNI), or inhibitors targeting PI3K, Akt or eNOS (LY294002, MK2206-2HCl or L-NAME, respectively). Furthermore, the expression levels of NLRP3, caspase-1, p-Akt, Akt, p-eNOS, and total eNOS were evaluated. Additionally, the production of reactive oxygen species, and levels of inflammatory factors, such as TNF-α, IL-1β, IL-6, IL-1 and adhesion molecules, such as ICAM-1, VCAM-1, P-selectin, and E-selectin were determined. The adherence rates of the neutrophils and monocytes were assessed as well. ResultsThe SP-induced hyperlipidemic cell model demonstrated increased expression of NLRP3 and caspase-1 proteins (P < 0.05) and elevated ROS levels (P < 0.01), and decreased phosphorylated-Akt and phosphorylated-eNOS expression (P < 0.05). In addition, SP significantly increased TNF-α, IL-1β, IL-6, ICAM-1, VCAM-1, P-selectin, and E-selectin levels (P < 0.01), decreased IL-10 levels (P < 0.01), and increased the adhesion rates of monocytes and neutrophils (P < 0.01). The SP-induced inflammatory response in HUVECs was ameliorated by κ-OR agonist, U50,488H. However, the protective effect of U50,488H was abolished by κ-OR antagonist, nor-BNI, and inhibitors of PI3K, Akt and eNOS. ConclusionOur findings suggest that κ-OR activation inhibits SP-induced inflammation by activating the PI3K/Akt/eNOS signaling pathway.

Inhibitory Effect of Paeonol on Apoptosis, Oxidative Stress, and Inflammatory Response in Human Umbilical Vein Endothelial Cells Induced by High Glucose and Palmitic Acid Induced Through Regulating SIRT1/FOXO3a/NF-κB Pathway.

Reactive oxygen species (ROS) induced by high glucose and high fat of diabetes mellitus (DM) finally caused the occurrence and progression of atherosclerosis and other macrovascular complications. Paeonol (Pae) exhibits anti-inflammation, antioxidation, and antiatherosclerosis activities. However, the role of Pae in diabetic cardiopathy has not been fully understood. Therefore, we aimed to investigate the role of Pae in diabetic cardiovascular diseases. Human umbilical vein endothelial cells (HUVECs) were exposed to high glucose and palmitic acid (HG/HP), a model DM environment and different doses of Pae. The viability and apoptotic rate of HUVECs were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry assay, respectively. Oxidative indicators (ROS, malondiadehyde [MDA], superoxide dismutase [SOD]), and inflammatory cytokines (tumor necrosis factor-α, interleukin-1β, and interleukin-6) were detected by 2,7-dichlorodihydrofluorescein diacetate, colorimetry, and enzyme-linked immunosorbent assay. The protein levels of Sirtuin type 1 (SIRT1), Bcl-2, Bax, Cleaved caspase-3, p-p65, and p-65 were detected by Western blot. The mRNA levels of Bcl-2 and Bax were detected by quantitative real-time polymerase chain reaction. The acetylation and protein levels of forkhead box O3a (FOXO3a) were detected by immunoprecipitation assay. SIRT1 silencing was used to confirm the role of Pae in the resistance to apoptosis, oxidative stress, and inflammatory response. Pae increased SIRT1 expression, cell viability, and SOD activity and suppressed apoptosis, the levels of p-p65/p-65, ROS, MDA, and inflammatory cytokines, and the expression of acetylated-FOXO3a induced by HG/HP in HUVECs. SIRT1 silencing abrogated the effect of Pae on HG/HP-mediated HUVECs. Inhibitory effect of Pae on apoptosis, oxidative stress, and inflammatory response in HUVECs induced by HG/HP induced through regulating SIRT1/FOXO3a/NF-κB pathway.

MicroRNA-17-3p suppresses NF-κB-mediated endothelial inflammation by targeting NIK and IKKβ binding protein.

Nuclear factor kappa B (NF-κB) activation contributes to many vascular inflammatory diseases. The present study tested the hypothesis that microRNA-17-3p (miR-17-3p) suppresses the pro-inflammatory responses via NF-κB signaling in vascular endothelium. Human umbilical vein endothelial cells (HUVECs), transfected with or without miR-17-3p agomir/antagomir, were exposed to lipopolysaccharide (LPS), and the inflammatory responses were determined. The cellular target of miR-17-3p was examined with dual-luciferase reporter assay. Mice were treated with miR-17-3p agomir and the degree of LPS-induced inflammation was determined. In HUVECs, LPS caused upregulation of miR-17-3p. Overexpression of miR-17-3p in HUVECs inhibited NIK and IKKβ binding protein (NIBP) protein expression and suppressed LPS-induced phosphorylation of inhibitor of kappa Bα (IκBα) and NF-κB-p65. The reduced NF-κB activity was paralleled by decreased protein levels of NF-κB-target gene products including pro-inflammatory cytokine [interleukin 6], chemokines [interleukin 8 and monocyte chemoattractant protein-1] and adhesion molecules [vascular cell adhesion molecule-1, intercellular adhesion molecule-1 and E-selectin]. Immunostaining revealed that overexpression of miR-17-3p reduced monocyte adhesion to LPS-stimulated endothelial cells. Inhibition of miR-17-3p with antagomir has the opposite effect on LPS-induced inflammatory responses in HUVECs. The anti-inflammatory effect of miR-17-3p was mimicked by NIBP knockdown. In mice treated with LPS, miR-17-3p expression was significantly increased. Systemic administration of miR-17-3p for 3 days suppressed LPS-induced NF-κB activation and monocyte adhesion to endothelium in lung tissues of the mice. In conclusion, miR-17-3p inhibits LPS-induced NF-κB activation in HUVECs by targeting NIBP. The findings therefore suggest that miR-17-3p is a potential therapeutic target/agent in the management of vascular inflammatory diseases.

SNHG1 Inhibits ox-LDL-Induced Inflammatory Response and Apoptosis of HUVECs via Up-Regulating GNAI2 and PCBP1.

Dysfunction of human endothelial cells is an important trigger for atherosclerosis. Oxidative low-density lipoprotein (ox-LDL) usually was used to stimulate the dysfunction of human umbilical vein endothelial cells (HUVECs). LncRNA SNHG1 (small nucleolar RNA host gene 1) is a cerebral infarction-associated gene. The present study was designed to investigate the role of SNHG1 in ox-LDL-induced HUVECs. Cell viability was evaluated by CCK-8 and MTT assay. Cell apoptosis was detected by flow cytometry analysis. Cell inflammatory response was evaluated by detecting LDH, IL-6, IL-1β levels. The results revealed that up-regulation of SNHG1 attenuated ox-LDL-induced cell injury and inflammatory response in HUVECs. Next, mechanism assays including RNA immunoprecipitation (RIP) assay, luciferase reporter assay, and RNA pull-down assay, helped us to identify the interaction between miR-556-5 and SNHG1. GNAI2 (G protein subunit alpha i2) and PCBP1 (poly(rC) binding protein 1) were identified as the downstream targets of miR-556-5p. SNHG1 regulated dysfunctions of ox-LDL-induced HUVECs via sponging miR-556-5p and up-regulating GNAI2 and PCBP1. SNHG1 attenuated cell injury and inflammatory response in ox-LDL-induced HUVECs via up-regulating both GNAI2 and PCBP1 at a miR-556-5p dependent way.

Inhibitory effects of aloin on TGFBIp-mediated septic responses

Aloin is the major anthraquinone glycoside obtained from the Aloe species. Transforming growth factor β-induced protein (TGFBIp) is an extracellular matrix protein and released by primary human umbilical vein endothelial cells (HUVECs) and functions as a mediator of experimental sepsis. We hypothesized that aloin could reduce TGFBIp-mediated severe inflammatory responses in HUVECs and mice. Aloin effectively inhibited lipopolysaccharide (LPS)-induced release of TGFBIp and suppressed TGFBIp-mediated septic responses. Aloin suppressed TGFBIp-induced sepsis lethality and pulmonary injury. Therefore, aloin is a potential therapeutic agent for various severe vascular inflammatory diseases, with inhibition of the TGFBIp signaling pathway as the mechanism of action.

PP2A alleviates oxidized LDL-induced endothelial dysfunction by regulating LOX-1/ROS/MAPK axis

AimsThe purpose of this study is to investigate the effect of PP2A on the progression of AS and the special molecular mechanism. Main methodsThe expression of PP2A in Human umbilical vein endothelial cells (HUVECs) induced by different concentrations of Ox-LDL was measured by RT-PCR and Western blot. The binding activity of PP2A and LOX-1 was determined by CoIP assay. Western blot was used to measure the protein expression of VCAM-1, ICAM-1 and MCP-1. Key findingThe results revealed that the expression of PP2A was decreased with the increase of Ox-LDL concentration in HUVECs. Overexpression of PP2A alleviated Ox-LDL-induced dysfunction and inflammatory response in HUVECs. The results of Co-immunoprecipitation (CoIP) showed that PP2A had direct effect on LOX-1, and PP2A inhibited the expression of LOX-1. In addition, overexpression of LOX-1 reversed the inhibitory effect of PP2A on Ox-LDL-induced dysfunction and inflammatory response in HUVECs. What is more, PP2A inhibited LOX-1/ROS/MAPK axis. Significanceit suggests that PP2A alleviates Ox-LDL-induced dysfunction and inflammatory response of HUVECs potentially by regulating the LOX-1/ROS/MAPK axis，which suggests that PP2A has anti-inflammatory effect during the formation of as, and the molecular therapy of PP2A provides a theoretical basis.

Apolipoprotein M and sphingosine-1-phosphate complex alleviates TNF-\u03b1-induced endothelial cell injury and inflammation through PI3K/AKT signaling pathway

BackgroundIn spite of the important role of Apolipoprotein-M (ApoM) and Sphingosine-1-Phosphate (S1P) played in atherosclerosis (AS), there was few related research reporting ApoM and S1P complex (ApoM-S1P) on biological activities of human umbilical vein endothelial cells (HUVECs). In this study, we explored the effect and mechanism of ApoM-S1P on TNF-α-induced inflammation in HUVECs.MethodsTNF-α was utilized to induce HUVEC injury and inflammation. After HUVECs were treated with antagonists of ApoM, S1P, ApoM + S1P, and ApoM + S1P + S1PR, calcein-acetoxymethyl ester was employed for the assessment of the adhesion of HUVECs to THP-1, immunofluorescence for the observation of caspase-1expression in HUVECs, reactive oxygen species (ROS) kit for the detection of ROS level in HUVECs. The impact of TNF-α, ApoM, S1P and S1PR antagonists on inflammatory response, pyroptosis and adhesion of THP-1 monocytes to HUVECs were determined by detecting expressions of pyroptosis related proteins (IL-1β, IL-18, ASC, NLRP3 and caspase-1), inflammatory cytokines (IL-6 and IL-10), adhesion molecules (E-selectin, ICAM-1, and VCAM-1) and p-PI3K/p-AKT by qRT-PCR and Western blot, as well as by ELISA.ResultsTNF-α could increase adhesion of THP-1 monocytes to HUVECs and induce inflammatory response and pyroptosis in HUVECs, indicated by up-regulated expressions of E-selectin, ICAM-1, VCAM-1, IL-1β, IL-18, caspase-1, ASC, NLRP3, and IL-6, and down-regulated expression of IL-10. Co-treatment of ApoM-S1P on TNF-α treated HUVECs could protect HUVECs from injury and inflammation, evidenced by the attenuation of expressions of pyroptosis related proteins, inflammatory cytokines, and adhesion molecules, as well as the augment of PI3K and AKT phosphorylation. JTE-013, an antagonist of S1PR2, could reverse the amelioration of ApoM-S1P on pyroptosis and inflammation of HUVECs, indicating that ApoM-S1P could bind to S1PR2 to protect HUVECs from injury and inflammation through activating PI3K/AKT pathway.ConclusionApoM-S1P could attenuate TNF-α induced injury and inflammatory response in HUVECs by binding to S1PR2 to activate PI3K/AKT pathway.

Legumain Promotes Atherosclerotic Vascular Remodeling.

Legumain, a recently discovered cysteine protease, is increased in both carotid plaques and plasma of patients with carotid atherosclerosis. Legumain increases the migration of human monocytes and human umbilical vein endothelial cells (HUVECs). However, the causal relationship between legumain and atherosclerosis formation is not clear. We assessed the expression of legumain in aortic atheromatous plaques and after wire-injury-induced femoral artery neointimal thickening and investigated the effect of chronic legumain infusion on atherogenesis in Apoe−/− mice. We also investigated the associated cellular and molecular mechanisms in vitro, by assessing the effects of legumain on inflammatory responses in HUVECs and THP-1 monocyte-derived macrophages; macrophage foam cell formation; and migration, proliferation, and extracellular matrix protein expression in human aortic smooth muscle cells (HASMCs). Legumain was expressed at high levels in atheromatous plaques and wire injury-induced neointimal lesions in Apoe−/− mice. Legumain was also expressed abundantly in THP-1 monocytes, THP-1 monocyte-derived macrophages, HASMCs, and HUVECs. Legumain suppressed lipopolysaccharide-induced mRNA expression of vascular cell adhesion molecule-1 (VCAM1), but potentiated the expression of interleukin-6 (IL6) and E-selectin (SELE) in HUVECs. Legumain enhanced the inflammatory M1 phenotype and oxidized low-density lipoprotein-induced foam cell formation in macrophages. Legumain did not alter the proliferation or apoptosis of HASMCs, but it increased their migration. Moreover, legumain increased the expression of collagen-3, fibronectin, and elastin, but not collagen-1, in HASMCs. Chronic infusion of legumain into Apoe−/− mice potentiated the development of atherosclerotic lesions, accompanied by vascular remodeling, an increase in the number of macrophages and ASMCs, and increased collagen-3 expression in plaques. Our study provides the first evidence that legumain contributes to the induction of atherosclerotic vascular remodeling.

Free fatty acids mediates human umbilical vein endothelial cells inflammation through toll-like receptor-4.

To investigate the role of Toll-like receptor-4 (TLR4) in the free fatty acids (FFAs) induced human umbilical vein endothelial cells (HUVECs) inflammation and to explore the underlying mechanisms. HUVECs and HEK293 cell lines were obtained from Shanghai Type Culture Collection. Cell counting kit-8 (CCK8) and flow cytometry (FCM) were performed to examine the cell viability and apoptosis rate of HUVECs induced by FFAs treatments with or without infection of toll-like receptor-4 interference (TLR4i) adenovirus. Enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the inflammatory cytokines release. Quantitative polymerase chain reaction (qPCR) and Western Blot (WB) were used to test the molecular mechanisms of inflammation. FFAs induced inflammatory responses in HUVECs via modulating the TLR4 receptor complex. TLR4i adenovirus interference increased cell viability and decreased cell apoptosis rate. FFAs treatments significantly increased the expressions of inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), C-C motif chemokine ligand 5 (CCL5) and CXC chemokine ligand 10 (CXCL10), while TLR4i adenovirus interference significantly reduced these cytokines levels. TLR4-mediated myeloid differential protein-88 (MyD88) expression activating the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and inhabiting kappa B kinase-beta (IKK-β). TLR4i adenovirus interference decreased the expressions of these genes at both mRNA level and protein level. TLR4 mediates FFAs induced inflammatory responses in HUVECs. TLR4 interference in HUVECs significantly reduces the inflammatory cytokines expression, decreases the cell apoptosis rate and increases cell viability.

HuR induces inflammatory responses in HUVECs and murine sepsis via binding to HMGB1.

The aim of the present study was to explore the roles of human antigen R (HuR) in sepsis. Reverse transcription‑quantitative polymerase chain reaction and western blot analyses demonstrated that overexpression of HuR increased the expression of high‑mobility group box 1 (HMGB1) in human umbilical vein endothelial cells (HUVECs). HMGB1 was investigated as a potential target of HuR through bioinformatics and RNA‑immunoprecipitation assays. Furthermore, treatment with HuR small interfering (si)RNA suppressed the lipopolysaccharide (LPS)‑mediated release of HMGB1 and reduced HMGB1‑mediated hyperpermeability and leukocyte migration in HUVECs and in septic mice. In addition, HuR‑siRNA injection reduced cecal ligation and puncture (CLP)‑induced HMGB1 release, reduced production of interleukin 6 and lowered mortality rates. Notably, the promotive effects of HuR overexpression on the inflammatory response were attenuated when HUVECs were co‑treated with HMGB1 short hairpin RNA. Therefore, the present results indicated that the ectopic expression of HuR may induce inflammatory responses and thus sepsis by activating the HMGB1 signaling pathway.

Salusin-α Attenuates Inflammatory Responses in Vascular Endothelial Cells.

Atherosclerosis accounts for numerous cardiovascular diseases, and cytokines have a critical role in acceleration or suppression of disease. Salusin-α presents a new class of bioactive peptides that can have anti-atherogenic properties. Therefore, the effects of salusin-α on the expression of some pro- and anti-inflammatory cytokines and on TNF-α-induced inflammatory responses in human umbilical vein endothelial cells (HUVECs) were examined. The involvement of the NF-κB pathway in effects of salusin-α in HUVECs was checked using Bay 11-7082 as an NF-κB inhibitor. The mRNA expression of pro-inflammatory cytokines including IL-6, IL-8, and IL-18 and anti-inflammatory cytokine IL-1Ra was assessed by real-time PCR. The protein levels of cytokines were measured by the ELISA method. Salusin-α suppressed both mRNA and protein expression of pro-inflammatory cytokines and induced mRNA and protein expression of IL-1Ra in HUVECs. Salusin-α suppressed TNF-α-induced inflammatory responses in HUVECs. The down-regulatory or up-regulatory effects of salusin-α on expression of cytokines could not be influenced by Bay 11-7082 pretreatment. Our findings indicate anti-inflammatory effects of salusin-α and suggest a novel peptide-based therapeutic strategy for atherosclerosis.

Pterostilbene protects against uraemia serum-induced endothelial cell damage via activation of Keap1/Nrf2/HO-1 signaling.

Chronic kidney disease causes uremia-related endothelial cell dysfunction associated with high risk for cardiovascular diseases. The vascular endothelium is permanently exposed to uraemic toxins including indoxyl sulfate, which provokes endothelial damage in subjects with end-stage renal disease. Pterostilbene (PT) is identified to be homologous derivative of resveratrol and exerts antioxidant and anti-inflammatory actions. However, the effects of PT on uraemic serum-induced endothelial cell damage have not been elucidated. In this study, we investigated the effects and mechanisms of PT on uraemic serum (US)-mediated injury in human umbilical vein endothelial cells (HUVECs). Treatment of US obviously reduced cell viability, inhibited superoxide dismutase activity and catalase activity, suppressed phosphorylated endothelial nitric oxide synthase (eNOS) protein level and eNOS activity, whereas promoted lactate dehydrogenase leakage, increased malondialdehyde, hydrogen peroxide, superoxide anions levels and NAD(P)H activity accompanied with increased nitrative stress and inflammatory response in HUVECs, and these changes were reversed after PT treatment. Under US environment, PT downregulated Kelch-like ECH-associated protein 1 (Keap1) and upregulated nuclear factor erythroid-2-related factor 2 (Nrf2) and its downstream target heme oxygenase-1 (HO-1) protein levels. Of note, the level of HO-1 was decreased after the transfection of cells with Nrf2-siRNA, and HO-1 inhibitor Snpp abolished the protective effects of PT on HUVECs in response to US. Collectively, our study demonstrated that PT is effective in reducing US-evoked endothelial cell dysfunction via suppression of oxidative/nitrative stress and inflammatory response, which at least partly depended on Keap1/Nrf2/HO-1 signaling pathway.

Felodipine inhibits ox-LDL-induced reactive oxygen species production and inflammation in human umbilical vein endothelial cells.

Oxidative stress and inflammation are involved in the pathogenesis of atherosclerosis. Calcium channel blockers (CCBs) inhibit the development of atherosclerosis, although the underlying molecular basis has not been completely elucidated. The present study was designed to investigate the effects of felodipine, a CCB, on inflammation and oxidative stress in human umbilical vein endothelial cells (HUVECs) and to examine the underlying mechanisms of action. Oxidized low‑density lipoprotein (ox‑LDL) was used to induce an inflammatory response in HUVECs. The effects of felodipine were investigated by measuring the content of nitric oxide (NO) and reactive oxygen species (ROS), the mRNA and protein levels of intercellular adhesion molecule 1 (ICAM‑1) and vascular cell adhesion protein 1 (VCAM‑1), and the mRNA levels of endothelial NO synthase (eNOS) and inducible NO synthase (iNOS), in addition to the adhesion ability of U937 cells to HUVECs. ROS and NO levels were significantly increased in HUVECs following 24‑h treatment with 25mg/l ox‑LDL (P<0.01). The increase in ROS was reversed by treatment with felodipine. In addition, NO levels were increased following treatment with 1µmol/l felodipine (P<0.05). The mRNA expression of ICAM‑1, VCAM‑1, eNOS and iNOS was increased (P<0.05). Administration of 0.1µM felodipine significantly decreased the expression of ICAM‑1, VCAM‑1, and iNOS (P<0.05). The number of U937 cells adhered to ox‑LDL‑treated HUVECs was significantly increased compared with control, which was reversed by felodipine (0.1µM). In conclusion, felodipine was demonstrated to inhibit oxidative stress and inflammatory responses, suggesting that it may be used to treat atherosclerosis.

Ghrelin suppresses inflammation in HUVECs by inhibiting ubiquitin-mediated uncoupling protein2 degradation.

Atherosclerosis is considered the major cause of heart attack, stroke and gangrene of the extremities, which is responsible for 50% of all mortality in Western countries. The pathogenesis and causes of atherosclerosis remain elusive. Recent studies highlight inflammation as a contributing factor for atherosclerosis in all stages of the disease process. In this study, we demonstrate that the treatment of human umbilical vein endothelial cells (HUVECs) with ghrelin inhibits the oxidized low-density lipoprotein (oxLDL)-induced inflammatory response, In addition, treatment with ghrelin led to the accumulation of uncoupling protein 2 (UCP2) in the cells, thus decreasing reactive oxygen species (ROS) generation. Moreover, the siRNA-mediated knockdown of UCP2 expression suggested that the inhibitory effects of ghrelin on the inflammatory response relied on its ability to induce the accumulation of cellular UCP2 levels. Further analysis indicated that the accumulation of UCP2 in the ghrelin-treated cells was due to the ability of ghrelin to inhibit the ubiquitination of UCP2 and prevent UCP2 degradation, resulting in the extended protein half-life of UCP2. On the whole, our data indicate that ghrelin inhibits the oxLDL-induced inflammatory response in HUVECs, and may thus have potential for use as an anti-atherosclerotic agent. Our data may also provide valuable insight into the pathogenesis of atherosclerosis.

The effects of endoplasmic reticulum stress inducer thapsigargin on the toxicity of ZnO or TiO2 nanoparticles to human endothelial cells

It was recently shown that ZnO nanoparticles (NPs) could induce endoplasmic reticulum (ER) stress in human umbilical vein endothelial cells (HUVECs). If ER stress is associated the toxicity of ZnO NPs, the presence of ER stress inducer thapsigargin (TG) should alter the response of HUVECs to ZnO NP exposure. In this study, we addressed this issue by assessing cytotoxicity, oxidative stress and inflammatory responses in ZnO NP exposed HUVECs with or without the presence of TG. Moreover, TiO2 NPs were used to compare the effects. Exposure to 32 μg/mL ZnO NPs (p < 0.05), but not TiO2 NPs (p > 0.05), significantly induced cytotoxicity as assessed by WST-1 and neutral red uptake assay, as well as intracellular ROS. ZnO NPs dose-dependently increased the accumulation of intracellular Zn ions, and ZnSO4 induced similar cytotoxic effects as ZnO NPs, which indicated a role of Zn ions. The release of inflammatory proteins tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) or the adhesion of THP-1 monocytes to HUVECs was not significantly affected by ZnO or TiO2 NP exposure (p > 0.05). The presence of 250 nM TG significantly induced cytotoxicity, release of IL-6 and THP-1 monocyte adhesion (p < 0.01), but did not significantly affect intracellular ROS or release of TNFα (p > 0.05). ANOVA analysis indicated no interaction between exposure to ZnO NPs and the presence of TG on almost all the endpoints (p > 0.05) except neutral red uptake assay (p < 0.01). We concluded ER stress is probably not associated with ZnO NP exposure induced oxidative stress and inflammatory responses in HUVECs.

Allicin Decreases Lipopolysaccharide-Induced Oxidative Stress and Inflammation in Human Umbilical Vein Endothelial Cells through Suppression of Mitochondrial Dysfunction and Activation of Nrf2

Background: Allicin, a major component of garlic, is regarded as a cardioprotective agent and is associated with increased endothelial function. Methods: The effects of allicin on lipopolysaccharide (LPS)-induced vascular oxidative stress and inflammation in cultured human umbilical vein endothelial cells (HUVECs) and the mechanisms underlying these effects were studied. The protective effects were measured using cell viability, a lactate dehydrogenase (LDH) assay and cell apoptosis as indicators, and the anti-oxidative activity was determined by measuring reactive oxygen species (ROS) generation, oxidative products and endogenous antioxidant enzyme activities. HUVEC mitochondrial function was assessed by determining mitochondrial membrane potential (MMP) collapse, cytochrome c production and mitochondrial ATP release. To investigate the potential underlying mechanisms, we also measured the expression of dynamic mitochondrial proteins using western blotting. Furthermore, we evaluated the Nrf2 antioxidant signaling pathway using an enzyme-linked immunosorbent assay (ELISA). Results: Our results demonstrated that allicin enhanced HUVEC proliferation, which was suppressed by LPS exposure, and LDH release. Allicin ameliorated LPS-induced apoptosis, suppressed ROS overproduction, reduced lipid peroxidation and decreased the endogenous antioxidant enzyme activities in HUVECs. These protective effects were associated with the inhibition of mitochondrial dysfunction as indicated by decreases in the MMP collapse, cytochrome c synthesis and mitochondrial ATP release. In addition, allicin attenuated the LPS-induced inflammatory responses, including endothelial cell adhesion and TNF-α and IL-8 production. Furthermore, allicin increased the expression of LXRα in a dose-dependent manner. Allicin-induced attenuation of inflammation was inhibited by LXRα siRNA treatment. Finally, allicin activated NF-E2-related factor 2 (Nrf2), which controls the defense against oxidative stress and inflammation. Conclusions: Taken together, the present data suggest that allicin attenuated the LPS-induced vascular injury process, which may be closely related to the oxidative stress and inflammatory response in HUVECs. Allicin modulated Nrf2 activation and protected the cells against LPS-induced vascular injury. Our findings suggest that allicin attenuated the LPS-induced inflammatory response in blood vessels.

Anti-inflammatory effects of pelargonidin on TGFBIp-induced responses.

Transforming growth factor β induced protein (TGFBIp) is an extracellular matrix protein expressed in several cell types in response to TGF-β. TGFBIp is released by human umbilical vein endothelial cells (HUVECs) and functions as a mediator of experimental sepsis. Pelargonidin (PEL) is a well-known red pigment found in plants, and has been reported as having important biological activities that are potentially beneficial for human health. This study was undertaken to investigate whether PEL can modulate TGFBIp-mediated inflammatory responses in HUVECs and in mice. The anti-inflammatory activities of PEL were determined by measuring permeability, leukocyte adhesion and migration, and activation of proinflammatory proteins in TGFBIp-activated HUVECs and mice. In addition, the beneficial effects of PEL on survival rate in a mouse sepsis model were tested. We found that PEL inhibited TGFBIp-induced barrier disruption, expression of cell adhesion molecules and adhesion/transendothelial migration of neutrophils to human endothelial cells. PEL also suppressed TGFBIp-induced hyperpermeability and leukocyte migration in vivo. These results suggest that PEL possesses anti-inflammatory properties that result in inhibition of hyperpermeability, expression of cell adhesion molecules, and adhesion and migration of leukocytes, thereby endorsing its usefulness as a therapy for vascular inflammatory diseases.