C -reactive protein induced expression of adhesion molecules in cultured cerebral microvascular endothelial cells

AIM lsehemic stroke can trigger an acute phase response resulting in a rise of plasma concentration of C - reactive protein （CRP）. Clinical data about the relationship between CRP and prognosis suggest that CRP might be involved in the pathogenesis of cerebral ischemia. To determine the possible role of CRP in ischemic stroke, we performed a dose - dependent experiment in mouse brain microvascular endothelial cells （bEnd. 3 cells） with emphasis on its relation to cell adhesions molecules.     

Monocyte adhesion induced by multi-walled carbon nanotubes and palmitic acid in endothelial cells and alveolar–endothelial co-cultures

Free palmitic acid (PA) is a potential pro-atherogenic stimulus that may aggravate particle-mediated cardiovascular health effects. We hypothesized that the presence of PA can aggravate oxidative stress and endothelial activation induced by multi-walled carbon nanotube (MWCNT) exposure in vitro. We investigated the interaction between direct exposure to MWCNTs and PA on THP-1 monocyte adhesion to human umbilical vein endothelial cells (HUVECs), as well as on indirect exposure in an alveolar–endothelial co-culture model with A549 cells and THP-1-derived macrophages exposed in inserts and the effect measured in the lower chamber on HUVECs and THP-1 cells. The exposure to MWCNTs, including a short (NM400) and long (NM402) type of entangled fibers, was associated with elevated levels of reactive oxygen species as well as a decrease in the intracellular glutathione concentration in HUVEC and A549 monocultures. Both effects were found to be independent of the presence of PA. MWCNT exposure significantly increased THP-1 monocyte adhesion to HUVECs, and co-exposure to PA aggravated the NM400-mediated adhesion but decreased the NM402-mediated adhesion. For the co-cultures, the exposure of A549 cells did not promote THP-1 adhesion to HUVECs in the lower chamber. When THP-1 macrophages were present on the cell culture inserts, there was a modest increase in the adhesion and an increase in interleukin-6 and interleukin-8 levels in the lower chamber whereas no tumor necrosis factor was detected. Overall, this study showed that direct exposure of HUVECs to MWCNTs was associated with oxidative stress and monocyte adhesion and the presence of PA increased the adhesion when exposed to NM400.     

The effects of caveolin - 1／eNOS pathway in monocyte adhesion to endothelial cells induced by oxidative stress

Leukocyte adhesion to endothelial cells is the initiate event of atherosclerosis, which includes injury of endothelial cells, leukocyte rolling, adhesion and extravasation. Many adhesion molecules such as E-selectin, P-selectin,the adhesion process.ICAM-1, VCAM, L-selectin, CD18, PECAM, VLA and ECM participate in Many factors such as infection of pathogenic organism,     

Stimulation of cyclic AMP production in human alveolar macrophages induced by inflammatory mediators and β-sympathicomimetics

We have investigated the effects of inflammatory mediators and β-adrenoceptor agonists on the adenylyl cyclase responsiveness in alveolar macrophages from control subjects, patients suffering from chronic obstructive pulmonary disease (COPD) and asthmatics. Basal cyclic AMP (cAMP) levels in alveolar macrophages from COPD patients were significantly elevated (plus 42%) as compared to controls. In addition, the adenylyl cyclase responsiveness to prostaglandin E₂, histamine and the β-adrenoceptor agonist salbutamol was significantly impaired in alveolar macrophages from COPD patients and asthmatics. The lipid mediator platelet activating factor showed no effect on cAMP production in all three alveolar macrophage populations. Furthermore, the cAMP-enhancing effects of isoprenaline, salbutamol and histamine appeared to be mediated via β₂-adrenoceptors and histamine H₂-receptor subtypes respectively. Taken together, these data suggest an intrinsic desensitization phenomenon in alveolar macrophages from COPD patients and asthmatics.     

Withaferin A inhibits tumor necrosis factor-α-induced expression of cell adhesion molecules by inactivation of Akt and NF-κB in human pulmonary epithelial cells

We here investigated the functional effect of withaferin A on airway inflammation and its action mechanism. Withaferin A inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human lung epithelial A549 cells stimulated with tumor necrosis factor-α (TNF-α), resulting in the suppression of leukocyte adhesion to lung epithelial A549 cells. In addition, withaferin A inhibited TNF-α-induced expression of adhesion molecules (ICAM-1 and VCAM-1) protein and mRNA in a dose-dependent manner. Withaferin A prevented DNA binding activity of nuclear factor-κB (NF-κB) and nuclear translocation of NF-κB. It also inhibited phosphorylation of Akt and extracellular signal-regulated kinase (ERK), which are upstream in the regulation of adhesion molecules by TNF-α. Furthermore, withaferin A inhibited U937 monocyte adhesion to A549 cells stimulated by TNF-α, suggesting that it may inhibit the binding of these cells by regulating the expression of critical adhesion molecules by TNF-α. Taken together, these results suggest that withaferin A inhibits cell adhesion through inhibition of ICAM-1 and VCAM-1 expression, at least in part, by blocking Akt and down-regulating NF-κB activity.     

Inhibition of LPS-induced NO production and NF-κB activation by a sesquiterpene fromSaussurea lappa

To elucidate the molecular mechanisms for the suppression of LPS-induced nitric oxide (NO) production by a dehydrocostus lactone (DL) from Saussurea lappa, we examined the preventive effect of this compound on NF-kappaB activation in LPS-treated RAW 264.7 macrophages and U937 human monocytic cells. The results suggest that the suppression of NO production is mediated by the inhibitory action on the i-NOS gene expression through the inactivation of NF-kappaB and this sesquiterpene lactone can act as a pharmacological inhibitor of the NF-kappaB activation.     

Effects of tanshinone Ⅱ-A sullfonate on adhesion molecule expression of endothelial cells and platelets in vitro

探讨丹参酮ⅡＡ磺酸钠（Ｔａｎ）对培养人脐静脉内皮细胞（ＨＵＶＥＣ）和人血小板表达粘附分子的影响．方法：用流动血细胞计数仪测定肿瘤坏死因子（ＴＮＦα）诱导人脐静脉内皮细胞ＩＣＡＭ１和凝血酶诱导人血小板Ｐ选择素的表达．结果：ＨＵＶＥＣ经ＴＮＦα处理后，明显增加细胞表面ＩＣＡＭ１的表达，增加ＨＬ６０细胞粘附到内皮细胞表面达加入细胞总数的３０％±６％（对照组为４６％±０７％）．在ＴＮＦα处理前，用Ｔａｎ（２５－２００μｍｏｌ·Ｌ－１）与ＨＵＶＥＣ共孵育，则Ｔａｎ剂量依赖性地抑制ＴＮＦα的作用．Ｔａｎ（２５－２００μｍｏｌ·Ｌ－１）与人血小板孵育后，可剂量依赖性地抑制凝血酶诱导人血小板表面Ｐｓｅｌｅｃｔｉｎ的表达．结论：Ｔａｎ可抑制内皮细胞和血小板表达粘附分子．     

Pyridoxine inhibits endothelial NOS uncoupling induced by oxidized low-density lipoprotein via the PKCα signalling pathway in human umbilical vein endothelial cells

BACKGROUND AND PURPOSE

One key mechanism for endothelial dysfunction is endothelial NOS (eNOS) uncoupling, whereby eNOS generates superoxide (O₂^•−) rather than NO. We explored the effect of pyridoxine on eNOS uncoupling induced by oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells (HUVECs) and the potential molecular mechanism.

EXPERIMENTAL APPROACH

HUVECs were incubated with ox-LDL with/without pyridoxine, N^G-nitro-L-arginine methylester (L-NAME), chelerythrine chloride (CHCI) or apocynin. Endothelial O₂^•− was measured using lucigenin chemiluminescence, and O₂^•−-sensitive fluorescent dye dihydroethidium (DHE). NO levels were measured by chemiluminescence, PepTag Assay for non-radioactive detection of PKC activity, depletion of PKCα and p47phox by siRNA silencing and the states of phospho-eNOS Thr495, total-eNOS, phospho-PKCα/βII, total PKC, phospho-PKCα, total PKCα and p47phox were measured by Western blot.

KEY RESULTS

Ox-LDL significantly increased O₂^•− production and reduced NO levels released from HUVECs; an effect reversed by eNOS inhibitor, L-NAME. Pyridoxine pretreatment significantly inhibited ox-LDL-induced O₂^•− generation and preserved NO levels. Pyridoxine also prevented the ox-LDL-induced reduction in phospho-eNOS Thr495 and PKC activity. These protective effects of pyridoxine were abolished by the PKC inhibitor, CHCI, or siRNA silencing of PKCα. However, depletion of p47phox or treatment with the NADPH oxidase inhibitor, apocynin, had no influence on these effects. Also, cytosol p47phox expression was unchanged by the different treatments.

CONCLUSIONS AND IMPLICATIONS

Pyridoxine mitigated eNOS uncoupling induced by ox-LDL. This protectant effect was related to phosphorylation of eNOS Thr495 stimulated by PKCα, not via NADPH oxidase. These results provide support for the use of pyridoxine in ox-LDL-related vascular endothelial dysfunction.     

TiO₂ nanoparticles induce dysfunction and activation of human endothelial cells

Nanoparticles can reach the blood and cause inflammation, suggesting that nanoparticles-endothelial cells interactions may be pathogenically relevant. We evaluated the effect of titanium dioxide nanoparticles (TiO?) on proliferation, death, and responses related with inflammatory processes such as monocytic adhesion and expression of adhesion molecules (E- and P-selectins, ICAM-1, VCAM-1, and PECAM-1) and with inflammatory molecules (tissue factor, angiotensin-II, VEGF, and oxidized LDL receptor-1) on human umbilical vein endothelial cells (HUVEC). We also evaluated the production of reactive oxygen species, nitric oxide production, and NF-κB pathway activation. Aggregates of TiO? of 300 nm or smaller and individual nanoparticles internalized into HUVEC inhibited proliferation strongly and induced apoptotic and necrotic death starting at 5 μg/cm2. Besides, TiO? induced activation of HUVEC through an increase in adhesion and in expression of adhesion molecules and other molecules involved with the inflammatory process. These effects were associated with oxidative stress and NF-κB pathway activation. In conclusion, TiO? induced HUVEC activation, inhibition of cell proliferation with increased cell death, and oxidative stress.     

Integrin β4 mAb inhibited apoptosis induced by deprivation of growth factors in vascular endothelial cells

INTRODUCTION The integrin α6β4 is a receptor for the lamininfamily of extracellular matrix proteins. In addition toserving a significant structural function in the assemblyof hemidesmosomes in epithelial cells[1,2], α6β4 pro-motes carcinoma cell migration and invasion[3,4]. Thediverse activities of this integrin are exemplified by itsability to trigger both the survival of keratinocytes andthe apoptosis of a number of carcinoma cell lines[4, . 5]These apparently contra…     

Apoptosis induced by α-anordrin in human promyelocytic leukemia HL-60 cells

Induction of apoptosis in human promyelocytic leukemia HL-60 cells by α-anordrin was estimated with the use of morphological and biochemical methods. α-Anordrin 2.5-50.0 μmol·L^-1 inhibited the growth of HL-60 cells in a dose-dependent manner and arrested them at G₁ phase of cell cycle. The cells exhibited marked morphological changes such as cell shrinkage, nuclear fragmentation, chromatin condensation. Agarose gel electrophoresis of DNA extracted from α-anordrin-treated cells revealed a “ladder” pattern. Nearly 63% of cells underwent apoptosis at α-anordrin concentration of 50 μmol·L^-1 as determined by flow cytometry. The data demonstrate that α-anordrin induces HL-60 cell apoptosis, which might be related to its antitumor action.     

2,3′,4,4′,5-Pentachlorobiphenyl impairs insulin-induced NO production partly through excessive ROS production in endothelial cells

Polychlorinated biphenyls (PCBs) have been reported to be associated with increased risk to hypertension, atherosclerosis, cardiovascular disease, etc. 2,3′,4,4′,5-Pentachlorobiphenyl, known as PCB-118, is a member of coplanar PCBs which renders their structure similar to polychlorinated dibenzo-p-dioxins (PCDDs) and has dioxin-like activity. In our current study, we investigated the effect of PCB-118 exposure on nitric oxide (NO) production and the underlying mechanisms in vitro. Exposure of PCB-118 impaired insulin-induced NO production and endothelial nitric oxide synthase (eNOS) activity in human umbilical vein endothelial cells (HUVECs) with no significant effect on cell viability. Furthermore, PCB-118 treatment induced oxidative stress. In addition, scavenging of reactive oxygen species (ROS) by 10?μM N-acetyl-l-cysteine (NAC) partly rescued impaired insulin-induced eNOS activities and NO productions induced by PCB-118 in HUVECs. Taken together, these results indicate that PCB-118 mediates lower eNOS activity and impairs insulin-induced NO production partly through excessive ROS production in endothelial cells.     

Inhibitory effect of quercetin on proliferation of human microvascular endothelial cells in vitro

AIM: To investigate the role of quercetin (Que) in the proliferation of cultured human skin microvascular endothelial cells (MVEC). METHODS: Cell count and [methyl-~3H]thymidine ([~3H]TdR) uptake assay were used to measure the effect of Que in the proliferation of cultured MVEC. Cytotoxicity of Que on MVEC was also evaluated by ~(51)Cr release assay. RESULTS: When MVEC were treated with Que, the proliferation was significantly inhibited in a time-course and dose-dependent manner. Que 5 μmol/L did not inhibit the proliferation of MVEC. When the concentration of Que increased to 20, 40, 80, and 160μmol/L, the cell numbers per well were decreased and the inhibition rate was 12.2％, 23.5％, 35.3％, and 54.1％ respectively with IC_(50) of 138 μmol/L. The inhibitory rate of [~3H]-TdR uptake was 18.7％, 34.4％, 48.9％, and 62.5％ respectively (IC_(50)=87.5 μmol/L). ~(51)Cr release assay showed that Que 160μmol/L incubated with MVEC from 1 to 16h had no clear cytotoxicity compared with control group. CONCL     

Aspirin protected against endothelial damage induced by LDL： role of endogenous NO synthase inhibitors in rats

AIM: To study the protective effect of aspirin on damages of the endothelium induced by low-density lipoprotein (LDL), and whether the protective effect of aspirin is related to reduction of nitric oxide synthase inhibitor level.METHODS: Vascular endothelial injury was induced by a single injection of native LDL (4 mg/kg) in rats. Vasodilator responses to acetylcholine (ACh) in the isolated aortic rings were determined, and serum concentrations ofasymmetric dimethylarginine (ADMA), malondialdehyde (MDA), tumour necrosis factor-α (TNF-α), and the activity of dimethylaminohydrolase (DDAH) were measured. RESULTS: A single injection of LDL (4 mg/kg)significantly decreased vasodilator responses to ACh, increased the serum level of ADMA, MDA, and TNF-α, anddecreased DDAH activity. Aspirin (30 or 100 mg/kg) markedly reduced the inhibition of vasodilator responses toACh by LDL, and the protective effect of aspirin at the lower dose was greater compared with high-dose aspiringroup. Aspirin inhibited the increased level of MDA and TNF-α induced by LDL. Aspirin at the dose of 30 mg/kg,but not at higher dose (100 mg/kg), significantly reduced the concentration of ADMA and increased the activity ofDDAH. CONCLUSION: Aspirin at the lower dose (30 mg/kg) protects the endothelium against damages elicitedby LDL in vivo, and the protective effect of aspirin on endothelium is related to reduction of ADMA concentrationby increasing DDAH activity.     

Enhanced serelaxin signalling in co‐cultures of human primary endothelial and smooth muscle cells

Background and Purpose

In the phase III clinical trial, RELAX‐AHF, serelaxin caused rapid and long‐lasting haemodynamic changes. However, the cellular mechanisms involved are unclear in humans.

Experimental Approach

This study examined the effects of serelaxin in co‐cultures of human primary endothelial cells (ECs) and smooth muscle cells (SMCs) on cAMP and cGMP signalling.

Key Results

Stimulation of HUVECs or human coronary artery endothelial cells (HCAECs) with serelaxin, concentration‐dependently increased cGMP accumulation in co‐cultured SMCs to a greater extent than in monocultures of either cell type. This was not observed in human umbilical artery endothelial cells (HUAECs) that do not express the relaxin receptor, RXFP1. Treatment of ECs with l‐N^G‐nitro arginine (NOARG; 30 μM, 30 min) inhibited serelaxin‐mediated (30 nM) cGMP accumulation in HUVECs, HCAECs and co‐cultured SMCs. In HCAECs, but not HUVECs, pre‐incubation with indomethacin (30 μM, 30 min) also inhibited cGMP accumulation in SMCs. Pre‐incubation of SMCs with the guanylate cyclase inhibitor ODQ (1 μM, 30 min) had no effect on serelaxin‐mediated (30 nM) cGMP accumulation in HUVECs and HCAECs but inhibited cGMP accumulation in SMCs. Serelaxin stimulation of HCAECs, but not HUVECs, increased cAMP accumulation concentration‐dependently in SMCs. Pre‐incubation of HCAECs with indomethacin, but not l‐NOARG, abolished cAMP accumulation in co‐cultured SMCs, suggesting involvement of prostanoids.

Conclusions and Implications

In co‐cultures, treatment of ECs with serelaxin caused marked cGMP accumulation in SMCs and with HCAEC also cAMP accumulation. Responses involved EC‐derived NO and with HCAEC prostanoid production. Thus, serelaxin differentially modulates vascular tone in different vascular beds.

Abbreviations

AHF

acute heart failure

DEA

diethylamine NONOate

ECs

endothelial cells

HCAEC

human coronary artery endothelial cell

HUAEC

human umbilical artery endothelial cell

HUASMC

human umbilical artery smooth muscle cell

HUVSMC

human umbilical vein smooth muscle cell

l‐NOARG

l‐N^G‐nitro arginine

SMCs

smooth muscle cells

     

Roles of ROS and PKC-βII in ionizing radiation-induced eNOS activation in human vascular endothelial cells

Vascular endothelial cells can absorb higher radiation doses than any other tissue in the body, and post-radiation impaired endothelial nitric oxide synthase (eNOS) function may be developed as a potential contributor to the pathogenesis of vascular injury. In this study, we investigated early alterations of eNOS signaling in human umbilical venous endothelial cells (HUVECs) exposed to X-ray radiation. We found that ionizing radiation increased eNOS phosphorylation at Ser-1177 and dephosphorylation at Thr-495 in HUVECs in a dose-dependent (≤ 20 Gy) and time-dependent (6–72 h) manner. The total expression levels of eNOS were unchanged by radiation. Although a transient but significant increase in extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation and a biphasic decline in Akt phosphorylation were observed after irradiation, these inhibitors were without effect on the radiation-induced changes in eNOS phosphorylation. There was an increase in protein kinase C-βII (PKC-βII) expression and the ablation of PKC-βII by small interfering RNA (siRNA) negated the radiation effect on the two eNOS phosphorylation events. Furthermore, when the radiation-induced increase in reactive oxygen species (ROS) generation was prevented by the anti-oxidant N-acetyl-l-cysteine, eNOS Ser-1177 phosphorylation and Thr-495 dephosphorylation in irradiated HUVECs were significantly reduced. However, transfection of PKC-β siRNA did not alter ROS production after irradiation, and NAC failed to block the radiation-induced increase in PKC-βII expression. Taken together, our results suggest that ionizing radiation-induced eNOS activation in human vascular endothelial cells is attributed to both the up-regulation of PKC-βII and the increase in ROS generation which were independent of each other.     

Effects of 17 β-estradiol on lipopolysacharride-induced intracellular adhesion molecule-1 mRNA expression and Ca2+ homeostasis alteration in human endothelial cells

Recent evidence showed that 17 β-estradiol (E₂) decreased cytokine-induced expression of cell adhesion molecules (CAM). Changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) has been shown to be associated with CAM expression in endothelial cells. Here, the effects of E₂ (1 μM, 24 h) on the expression of intracellular adhesion molecule-1 (ICAM-1) and [Ca²⁺]_i were investigated in a lipopolysaccharide (LPS) (100 ng/mL, 18 h)-stimulated human endothelial cell line, EA.hy926, using real-time PCR and spectrofluorometry, respectively. PCR analysis revealed a significant increase in ICAM-1 expression in calcium ionophore A23187 (1 nM)- or LPS-stimulated cells. Pretreatment of cells with E₂ significantly inhibited LPS-induced ICAM-1 mRNA expression. [Ca²⁺]_i was monitored in Fura-2 AM-loaded cells in the presence and absence of extracellular Ca²⁺ with thapsigargin (TG, 1 μM), a sarco/endoplasmic reticulum ATPase inhibitor or ATP (100 μM). The extent of TG- or ATP-induced [Ca²⁺]_i increase was significantly higher in LPS-stimulated cells than in control cells. Pre-treatment of LPS-stimulated cells with E₂ limited the Ca²⁺ response to the same level as in control cells. Furthermore, ICI 182,780, an estrogen receptor antagonist, attenuated the inhibitory actions of E₂ on ICAM-1 mRNA expression and Ca²⁺ responses, suggesting that estrogen receptors mediate, at least in part, the effects of estrogen. These data suggest a potential underlying mechanism for the protective effect of E₂ against atherosclerosis.