Nicotine stimulates adhesion molecular expression via calcium influx and mitogen-activated protein kinases in human endothelial cells

To evaluate the effect of nicotine on endothelium dysfunction and development of vascular diseases, we investigated the influence on adhesion molecular expression mediated by nicotine and the mechanism of this effect in human umbilical vein endothelial cells (HUVECs). The result showed that nicotine could induce surface/soluble vascular cell adhesion molecule (VCAM-1) and endothelial selectin (E-selectin) expression in a time-response decline manner and the peak appeared at 15 min. This action could be mediated by mitogen-activated protein kinase/extracellular signal regulated kinase 1/2 (MAPK/ERK1/2) and MAPK/p38 because their activation could be distinctly blocked by MAPK inhibitors, PD098059 or SB203580. Mecamylamine (non-selective nicotinic receptor inhibitor), alpha-bungarotoxin (alpha7 nicotinic receptor inhibitor) could block Ca2+ accumulation, and then, prevented the phosphorylation on ERK1/2 and p38. They also inhibited the surface/soluble VCAM-1, E-selectin production of HUVECs modulated by nicotine. Therefore, we concluded that: (i) nicotine obviously up-regulates VCAM-1 and E-selectin expression at 15 min in HUVECs, (ii) nicotine activates HUVECs triggered by the ERK1/2 and p38 phosphorylation with an involvement of intracellular calcium mobilization chiefly mediated by alpha7 nicotinic receptor, (iii) intracellular Ca2+ activates a sequential pathway from alpha7 nicotinic receptor to the phosphorylation of ERK1/2, p38. These elucidate that nicotine activates HUVECs through fast signal transduction pathway and arguments their capacity of adhesion molecular production. Further more nicotine may contribute its influence to the progression of vascular disease such as atherosclerotic lesion.     

Selenoprotein S protects against high glucose-induced vascular endothelial apoptosis through the PKCβII/JNK/Bcl-2 pathway

Vascular endothelial apoptosis is closely associated with the pathogenesis and progression of diabetic macrovascular diseases. Selenoprotein S (SelS) participates in the protection of vascular endothelial and smooth muscle cells from oxidative and endoplasmic reticulum stress-induced injury. However, whether SelS can protect vascular endothelium from high glucose (HG)-induced apoptosis and the underlying mechanism remains unclear. The present study preliminarily analyzed aortic endothelial apoptosis and SelS expression in diabetic rats in vivo and the effects of HG on human umbilical vein endothelial cell (HUVEC) apoptosis and SelS expression in vitro. Subsequently, SelS expression was up- or downregulated in HUVECs using the pcDNA3.1-SelS recombinant plasmid and SelS-specific small interfering RNAs, and the effects of high/low SelS expression on HG-induced HUVEC apoptosis and a possible molecular mechanism were analyzed. As expected, HG induced vascular endothelial apoptosis and upregulated endothelial SelS expression in vivo and in vitro. SelS overexpression in HUVECs suppressed HG-induced increase in apoptosis and cleaved caspase3 level, accompanied by reduced protein kinase CβII (PKCβII), c-JUN N-terminal kinase (JNK), and B-cell lymphoma/leukemia-2 (Bcl-2) phosphorylation. In contrast, inhibiting SelS expression in HUVECs further aggravated HG-induced increase in apoptosis and cleaved caspase3 level, which was accompanied by increased PKCβII, JNK, and Bcl-2 phosphorylation. Pretreatment with PKC activators blocked the protective effects of SelS and increased the apoptosis and cleaved caspase3 level in HUVECs. In summary, SelS protects vascular endothelium from HG-induced apoptosis, and this was achieved through the inhibition of PKCβII/JNK/Bcl-2 pathway to eventually inhibit caspase3 activation. SelS may be a promising target for the prevention and treatment of diabetic macrovascular complications.     

Regulation of MAP kinase-mediated endothelial dysfunction in hyperglycemia via arginase I and eNOS dysregulation

Emerging evidence suggests that arginase contributes to endothelial dysfunction in diabetes. Intracellular signaling pathways, which interplay between arginase and eNOS enzyme activity leading to the development of endothelial dysfunction in hyperglycemia are not fully understood. Here, we analyzed the possible involvement of hyperglycemia (HG) induced arginase expression in eNOS protein regulation and activity and also the impact of arginase inhibition on eNOS activity. Furthermore, the roles of p38 MAPK and Erk1/2 phosphorylation in upregulation of arginase expression and eNOS dysregulation in endothelial cells (ECs) under hyperglycemia were evaluated. Protein analysis showed a concurrent increase in arginase I expression and decrease in eNOS expression and phosphorylation at Ser¹¹⁷⁷ under HG conditions. There was no simultaneous change in phosphorylation of eNOS at Thr⁴⁹⁵ in HG. Arginase inhibition prevented increased arginase activity, restored impaired NO bioavailability and reduced superoxide anion generation. Inhibition of MAP-kinases demonstrated that, unlike Erk1/2, p38 MAPK is an upstream activator in a signaling cascade leading to increased arginase I in HG conditions. P38 MAPK protein expression and phosphorylation were increased in response to HG. In the presence of a p38 MAPK inhibitor, HG-induced arginase expression was blunted. Although Erk1/2 was activated in HG, increased arginase expression was not blocked by co-treatment with an Erk1/2 inhibitor. Activation of both, p38 MAPK and Erk1/2 in HG, induced a downregulation in eNOS activity. Hence, applying MAPK inhibitors increased eNOS phosphorylation in HG.In conclusion, these findings demonstrate contributions of arginase I in the development of endothelial cell dysfunction under HG conditions via impaired eNOS regulation, which maybe mediated by p38 MAPK.     

p38 MAPK pathway is involved in high glucose-induced thioredoxin interacting protein induction in mouse mesangial cells

Excessive reactive oxygen species (ROS) play a key role in the pathogenesis of diabetic nephropathy. The thioredoxin (TRX) system, a major thiol antioxidant system, regulates the reduction of intracellular ROS. Here we show that high glucose (HG) inhibits TRX ROS-scavenging function through p38 mitogen-activated protein kinase (MAPK)-mediated induction of thioredoxin interacting protein (TXNIP) in mouse mesangial cells (MMCs). Knockdown of TXNIP in MMCs reversed HG-induced reduction of TRX activity and inhibited HG-induced activation of p38 MAPK and increased synthesis of TGF-β1 and fibronectin. These data suggest that HG-induced overexpression of TXNIP in MMCs, which may be via the p38 MAPK pathway.     

Apollon/Bruce is upregulated by Humanin

Hydrogen sulfide (H₂S) protects cardiomyoblasts against high glucose (HG)-induced injury by inhibiting the activation of p38 mitogen-activated protein kinase (MAPK). This study aims to determine whether the leptin–p38 MAPK pathway is involved in HG-induced injury and whether exogenous H₂S prevents the HG-induced insult through inhibition of the leptin–p38 MAPK pathway in H9c2 cells. H9c2 cells were treated with 35 mM glucose (HG) for 24 h to establish a HG-induced cardiomyocyte injury model. Cell viability; mitochondrial membrane potential (ΔΨ _m); apoptosis; reactive oxygen species (ROS) level; and leptin, leptin receptor, and p38 MAPK expression level were measured by the methods indicated. The results showed pretreatment of H9c2 cells with NaHS before exposure to HG led to an increase in cell viability, decrease in apoptotic cells, ROS generation, and a loss of ΔΨ _m. Exposure of H9c2 cells to 35 mM glucose for 24 h significantly upregulated the expression levels of leptin and leptin receptors. The increased expression levels of leptin and leptin receptors were markedly attenuated by pretreatment with 400 μM NaHS. In addition, the HG-induced increase in phosphorylated (p) p38 MAPK expression was ameliorated by pretreatment with 50 ng/ml leptin antagonist. In conclusion, the present study has demonstrated for the first time that the leptin–p38 MAPK pathway contributes to the HG-induced injury in H9c2 cells and that exogenous H₂S protects H9c2 cells against HG-induced injury at least in part by inhibiting the activation of leptin–p38 MAPK pathway.     

The p38 mitogen-activated protein kinase mediates cytoskeletal remodeling in pulmonary microvascular endothelial cells upon intracellular adhesion molecule-1 ligation

Changes in the cytoskeleton of endothelial cells (ECs) play important roles in mediating neutrophil migration during inflammation. Previous studies demonstrated that neutrophil adherence to TNF-alpha-treated pulmonary microvascular ECs induced cytoskeletal remodeling in ECs that required ICAM-1 ligation and oxidant production and was mimicked by cross-linking ICAM-1. In this study, we examined the role of ICAM-1-induced signaling pathways in mediating actin cytoskeletal remodeling. Cross-linking ICAM-1 induced alterations in ICAM-1 distribution, as well as the filamentous actin rearrangements and stiffening of ECs shown previously. ICAM-1 cross-linking induced phosphorylation of the p38 mitogen-activated protein kinase (MAPK) that was inhibited by allopurinol and also induced an increase in the activity of the p38 MAPK that was inhibited by SB203580. However, SB203580 had no effect on oxidant production in ECs or ICAM-1 clustering. ICAM-1 cross-linking also induced phosphorylation of heat shock protein 27, an actin-binding protein that may be involved in filamentous actin polymerization. The time course of heat shock protein 27 phosphorylation paralleled that of p38 MAPK phosphorylation and was completely inhibited by SB203580. In addition, SB203580 blocked the EC stiffening response induced by either neutrophil adherence or ICAM-1 cross-linking. Moreover, pretreatment of ECs with SB203580 reduced neutrophil migration toward EC junctions. Taken together, these data demonstrate that activation of p38 MAPK, mediated by xanthine oxidase-generated oxidant production, is required for cytoskeletal remodeling in ECs induced by ICAM-1 cross-linking or neutrophil adherence. These cytoskeletal changes in ECs may in turn modulate neutrophil migration toward EC junctions.     

Inhibition of NADPH oxidase-related oxidative stress-triggered signaling by honokiol suppresses high glucose-induced human endothelial cell apoptosis

Angiopathy is a major complication of diabetes. Abnormally high blood glucose is a crucial risk factor for endothelial cell damage. Nuclear factor-kappaB (NF-kappaB) has been demonstrated as a mediated signaling in hyperglycemia or oxidative stress-triggered apoptosis of endothelial cells. Here we explored the efficacy of honokiol, a small molecular weight natural product, on NADPH oxidase-related oxidative stress-mediated NF-kappaB-regulated signaling and apoptosis in human umbilical vein endothelial cells (HUVECs) under hyperglycemic conditions. The methods of morphological Hoechst staining and annexin V/propidium iodide staining were used to detect apoptosis. Submicromolar concentrations of honokiol suppressed the increases of NADPH oxidase activity, Rac-1 phosphorylation, p22(phox) protein expression, and reactive oxygen species production in high glucose (HG)-stimulated HUVECs. The degradation of IkappaBalpha and increase of NF-kappaB activity were inhibited by honokiol in HG-treated HUVECs. Moreover, honokiol (0.125-1 microM) also suppressed HG-induced cyclooxygenase (COX)-2 upregulation and prostaglandin E(2) production in HUVECs. Honokiol could reduce increased caspase-3 activity and the subsequent apoptosis and cell death triggered by HG. These results imply that inhibition of NADPH oxidase-related oxidative stress by honokiol suppresses the HG-induced NF-kappaB-regulated COX-2 upregulation, apoptosis, and cell death in HUVECs, which has the potential to be developed as a therapeutic agent to prevent hyperglycemia-induced endothelial damage.     

Basic fibroblast growth factor inhibits radiation-induced apoptosis of HUVECs. II. The RAS/MAPK pathway and phosphorylation of BAD at serine 112

Radiation-induced endothelial cell apoptosis is involved in the development of many radiation injuries, including radiation-induced skin ulcers. The proangiogenic growth factor basic fibroblast growth factor (bFGF, NUDT6) enhances endothelial cell survival. In the present study, we set up a model of apoptosis in which primary cultured human umbilical vein endothelial cells (HUVECs) were irradiated with (60)Co gamma rays to explore the effects of bFGF on radiation-induced apoptosis of HUVECs and the signaling pathways involved. We found that bFGF inhibited radiation-induced apoptosis of HUVECs, and that the effect was mediated in part by the RAS/MEK/ MAPK/RSK (p90 ribosomal S6 kinase)/BAD pathway. This pathway was activated by exposure of irradiated HUVECs to bFGF, involving phosphorylation of FGFR, MEK and p44/42 MAPK. The survival-enhancing effect of bFGF was partly inhibited by U0126 and PD98059. The fact that the anti-apoptosis effect of bFGF on irradiated HUVECs was not completely abrogated by U0126 and PD98059 suggests that other survival signaling pathways may exist. Transfection of a dominant-negative form of RSK2 (DN RSK2) partly blocked the anti-apoptosis effect of bFGF in irradiated HUVECs. Moreover, we provide evidence for the first time that bFGF induced BAD phosphorylation (at serine 112) and CREB (cAMP response element-binding protein) activation (phosphorylation at serine 133) in gamma-irradiated HUVECs. In our model, inhibition of MAPK signaling-dependent phosphorylation of BAD at serine 112 promoted increased association with BCL-X(L), suggesting that MAPK pathway-dependent serine 112 phosphorylation of BAD is critical for the effect of bFGF on cell survival. These results showed that RAS/MAPK/BAD pathway participated in the bFGF-induced effect on survival of HUVECs exposed to radiation. It is suggested that RAS/ MAPK pathway in tumor vascular endothelium could be a potential therapeutic target to enhance the efficacy of ionizing radiation.     

Heterogeneity of the signal transduction pathways for VEGF-induced MAPKs activation in human vascular endothelial cells.

Vascular endothelial growth factor (VEGF) activates ERK and p38 MAPK in endothelial cells (ECs). The present study was aimed to compare its intracellular signal transduction pathways between three primary cultures of human ECs including human aortic ECs (HAECs), human umbilical vein ECs (HUVECs), and human microvascular ECs (HMVECs). VEGF activated ERK and p38 MAPK in all of three ECs. Isoforms of p38 MAPK that were activated by VEGF in HUVECs were p38-alpha and p38-delta. GF109203X, a specific inhibitor of PKC, markedly inhibited VEGF-induced activation of ERK and p38 MAPK in HAECs and HUVECs, whereas it exhibited little effect in HMVECs. In contrast, dominant negative mutant of Ha-Ras almost completely abrogated VEGF-induced activation of ERK and p38 MAPK in HMVECs. Although dominant negative mutant of Ha-Ras substantially inhibited the basal activities of ERK and p38 MAPK, it exhibited marginal effect on VEGF-induced activation of ERK and p38 MAPK in HUVECs and HAECs. The activation of Ras by VEGF appeared to be most prominent in HMVECs. These results indicate that intracellular signal transduction pathways for VEGF-induced activation of MAPKs are heterogeneous and vary depending on the origin of ECs.Copyright 2001 Wiley-Liss, Inc.     

Unfractionated heparin inhibits lipopolysaccharide-induced inflammatory response through blocking p38 MAPK and NF-κB activation on endothelial cell

Heparins, including unfractionated heparin (UFH) and low-molecular-weight heparins (LMWH), are glycosaminoglycans that are largely used as anti-thrombotic drugs. While the mechanisms of their anticoagulant actions in blood have been extensively studied, their effects on the inflammation of the endothelium are still under investigation since the endothelium plays a central role in sepsis. Furthermore, UFH is much cheaper than LMWH. The aim of this study was to determine how UFH regulates lipopolysaccharide (LPS)-induced inflammatory response on endothelial cells in vitro, and define the role of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) in mediating this effect. Human pulmonary microvascular endothelial cells (HPMECs) were pretreated with UFH (0.01U/ml-10U/ml), prior to stimulation with LPS (10μg/ml). Markers of systemic inflammation and endothelial activation were assessed. Interleukin (IL)-1β, IL-6, E-selectin, intercellular adhesion molecule (ICAM)-1 release were subsequently measured at 2h, 6h and 12h. Phosphorylation of p38 MAPK at 2h, 6h and nuclear translocation of the proinflammatory NF-κB at 2h were assessed. In HPMEC, UFH significantly attenuated LPS-induced production of IL-1β, IL-6, E-selectin and ICAM-1, as well as phosphorylation of p38 MAPK and NF-κB translocation, especially in 10U/ml. In conclusion, UFH at high dose significantly protects against endothelial-cell-mediated immune response. The inhibition of p38 MAPK and NF-κB activation certainly represents one of the mechanisms by which UFH exerts its anti-inflammatory effect.     

Atorvastatin suppresses LPS-induced rapid upregulation of Toll-like receptor 4 and its signaling pathway in endothelial cells

In the present study, we tested our hypothesis that atorvastatin exerts its anti-inflammation effect via suppressing LPS-induced rapid upregulation of Toll-like receptor 4 (TLR4) mRNA and its downstream p38, ERK, and NF-κB signaling pathways in human umbilical-vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs). TLR4 mRNA expression and its downstream kinase activities induced by LPS alone or atorvastatin + LPS in endothelial cells were quantified using quantitative real-time PCR and enzyme-linked immunosorbent assay. Preincubation of LPS-stimulated endothelial cells with TLR4 siRNA was conducted to identify the target of the anti-inflammatory effects of atorvastatin. Atorvastatin incubation resulted in the reduction of LPS-induced TLR4 mRNA expression, ERK1/2 and P38 MAPK phosphorylation, and NF-κB binding activity. Pretreatment with MEK/ERK1/2 inhibitor PD98059 attenuated atorvastatin + LPS-induced NF-κB activity but had no effect on P38 MAPK phosphorylation. In contrast, pretreatment with P38 MAPK inhibitor SB203580 resulted in upregulation of atorvastatin + LPS-induced ERK1/2 phosphorylation but had no significant effects on NF-κB activity. On the other hand, blocking NF-κB with SN50 produced no effects on atorvastatin + LPS-induced ERK1/2 and P38 MAPK phosphorylation. Moreover, TLR4 gene silencing produced the same effects as the atorvastatin treatment. In conclusion, atorvastatin downregulated TLR4 mRNA expression by two distinct signaling pathways. First, atorvastatin stabilized Iκ-Bα, which directly inhibited NF-κB activation. Second, atorvastatin inactivated ERK phosphorylation, which indirectly inhibited NF-κB activation. Suppression of p38 MAPK by atorvastatin upregulates ERK but exerts no effect on NF-κB.     

Involvement of integrins, MAPK, and NF-kappaB in regulation of the shear stress-induced MMP-9 expression in endothelial cells

Variations in the matrix metalloproteinase (MMP)-9 gene are related to the presence and severity of atherosclerosis. The aim of this study was to determine the signaling pathways of MMP-9 in endothelial cells subjected to low fluid shear stress. We found that low fluid shear stress significantly increased MMP-9 expression, IkappaBalpha degradation, NF-kappaB DNA-binding activity and phosphorylation of MAPK in cultured human umbilical vein endothelial cells (HUVECs). Inhibition of NF-kappaB resulted in remarkable downregulation of stress-induced MMP-9 expression. Pretreatment of HUVECs with inhibitors of p38 mitogen-activating protein kinase (MAPK) and extracellular signal-regulated kinase1/2 (ERK1/2) also led to significant suppression of stress-induced MMP-9 expression and NF-kappaB DNA-binding activity. Similarly, addition of integrins inhibitor to HUVECs suppressed the stress-induced MMP-9 expression, IkappaBalpha degradation, NF-kappaB DNA-binding activity and the phosphorylation of p38 MAPK, ERK1/2. Our findings demonstrated that the shear stress-induced MMP-9 expression involved integrins-p38 MAPK or ERK1/2-NF-kappaB signaling pathways.     

Selenium inhibits high glucose-induced cyclooxygenase-2 and P-selectin expression in vascular endothelial cells

Selenium as a component of glutathione peroxidase may be beneficial in insulin resistance, hence potentially may modify the risk of diabetes and cardiovascular disease. The aim of our study was to evaluate whether selenium can also alter high glucose (HG), advanced glycation end products (AGE), high insulin (HI) and H₂O₂-induced expression of cyclooxygenase (COX)-2 and P-selectin. Human umbilical vein endothelial cells (HUVECs) were pretreated with selenium and stimulated by HG, AGE, HI and H₂O₂. Selenium significantly inhibited HG, AGE, HI and H₂O₂-induced expression of COX-2 and P-selectin. Moreover, selenium also inhibited HG, AGE, HI and H₂O₂-induced activation of p38 mitogen-activated protein kinase (p38 MAPK), which indicated that the preventive effects of selenium on COX-2 and P-selectin may be associated with p38. Our results indicated that selenium supplementation can reduce HG, AGE, HI and H₂O₂-induced expression of COX-2 and P-selectin by inhibition of the p38 pathway.     

Involvement of p38 MAPK pathway in GLP-1-induced inhibition of apoptosis in human umbilical vein endothelial cells

The aim of the present study was to investigate the effect of glucagon-like peptide-1 (GLP-1) on palmitate-induced apoptosis of human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. HUVECs were cultured in vitro, and then treated by palmitate to induce apoptosis. Meanwhile, GLP-1 was added to explore its effect. After 24 h of the treatments, Caspase-3 activity and DNA fragmentation were measured using ELISA kits. Phospho-p38 mitogen-activated protein kinase (p-p38 MAPK) expression was detected by Western blot. The results showed that incubating HUVECs with 0.125 mmol/L GLP-1 increased Caspase-3 activity and DNA fragmentation. GLP-1 significantly inhibited palmitate-induced increases of Caspase-3 activity and DNA fragmentation in a concentration-dependent manner. Moreover, GLP-1 inhibited the up-regulation of p-p38 MAPK expression induced by palmitate in HUVECs. These results suggest GLP-1 protects HUVECs against lipo-apoptosis, and this effect may be mediated through inhibiting p38 MAPK pathway.