Adhesion Of Monocytic THP-1 Cells Research Articles

The Shear Stress-Regulated Expression of Glypican-4 in Endothelial Dysfunction In Vitro and Its Clinical Significance in Atherosclerosis.

Retention of circulating lipoproteins by their interaction with extracellular matrix molecules has been suggested as an underlying mechanism for atherosclerosis. We investigated the role of glypican-4 (GPC4), a heparan sulfate (HS) proteoglycan, in the development of endothelial dysfunction and plaque progression; Expression of GPC4 and HS was investigated in human umbilical vein/artery endothelial cells (HUVECs/HUAECs) using flow cytometry, qPCR, and immunofluorescent staining. Leukocyte adhesion was determined in HUVECs in bifurcation chamber slides under dynamic flow. The association between the degree of inflammation and GPC4, HS, and syndecan-4 expressions was analyzed in human carotid plaques; GPC4 was expressed in HUVECs/HUAECs. In HUVECs, GPC4 protein expression was higher in laminar than in non-uniform shear stress regions after a 1-day or 10-day flow (p < 0.01 each). The HS expression was higher under laminar flow after a 1 day (p < 0.001). Monocytic THP-1 cell adhesion to HUVECs was facilitated by GPC4 knock-down (p < 0.001) without affecting adhesion molecule expression. GPC4 and HS expression was lower in more-inflamed than in less-inflamed plaque shoulders (p < 0.05, each), especially in vulnerable plaque sections; Reduced expression of GPC4 was associated with atherogenic conditions, suggesting the involvement of GPC4 in both early and advanced stages of atherosclerosis.

EPSTI1 promotes monocyte adhesion to endothelial cells in vitro via upregulating VCAM-1 and ICAM-1 expression.

Atherosclerosis is a chronic inflammatory disease of arterial wall, and circulating monocyte adhesion to endothelial cells is a crucial step in the pathogenesis of atherosclerosis. Epithelial-stromal interaction 1 (EPSTI1) is a novel gene, which is dramatically induced by epithelial-stromal interaction in human breast cancer. EPSTI1 expression is not only restricted to the breast but also in other normal tissues. In this study we investigated the role of EPSTI1 in monocyte-endothelial cell adhesion and its expression pattern in atherosclerotic plaques. We showed that EPSTI1 was dramatically upregulated in human and mouse atherosclerotic plaques when compared with normal arteries. In addition, the expression of EPSTI1 in endothelial cells of human and mouse atherosclerotic plaques is significantly higher than that of the normal arteries. Furthermore, we demonstrated that EPSTI1 promoted human monocytic THP-1 cell adhesion to human umbilical vein endothelial cells (HUVECs) via upregulating VCAM-1 and ICAM-1 expression in HUVECs. Treatment with LPS (100, 500, 1000 ng/mL) induced EPSTI1 expression in HUVECs at both mRNA and protein levels in a dose- and time-dependent manner. Knockdown of EPSTI1 significantly inhibited LPS-induced monocyte-endothelial cell adhesion via downregulation of VCAM-1 and ICAM-1. Moreover, we revealed that LPS induced EPSTI1 expression through p65 nuclear translocation. Thus, we conclude that EPSTI1 promotes THP-1 cell adhesion to endothelial cells by upregulating VCAM-1 and ICAM-1 expression, implying its potential role in the development of atherosclerosis.

Role of protein tyrosine phosphatase 1B (PTP1B) in the increased sensitivity of endothelial cells to a promigratory effect of erythropoietin in an inflammatory environment.

The proliferation and migration of endothelial cells are vascular events of inflammation, a process which can also potentiate the effects of promigratory factors. With the aim of investigating possible modifications in the activity of erythropoietin (Epo) in an inflammatory environment, we found that Epo at a non-promigratory concentration was capable of stimulating EA.hy926 endothelial cell migration when TNF-α was present. VCAM-1 and ICAM-1 expression, as well as adhesion of monocytic THP-1 cells to endothelial layers were also increased. Structurally modified Epo (carbamylation or N-homocysteinylation) did not exhibit these effects. The sensitizing effect of TNF-α on Epo activity was mediated by the Epo receptor. Inhibition assays targeting the PI3K/mTOR/NF-κB pathway, shared by Epo and TNF-α, show a cross-talk between both cytokines. As observed in assays using antioxidants, cell migration elicited by TNF-α+Epo depended on TNF-α-generated reactive oxygen species (ROS). ROS-mediated inactivation of protein tyrosine phosphatase 1B (PTP1B), involved in Epo signaling termination, could explain the synergistic effect of these cytokines. Our results suggest that ROS generated by inflammation inactivate PTP1B, causing the Epo signal to last longer. This mechanism, along with the cross-talk between both cytokines, could explain the sensitizing action of TNF-α on the migratory effect of Epo.

LOX-1 Regulates P. gingivalis-Induced Monocyte Migration and Adhesion to Human Umbilical Vein Endothelial Cells.

Porphyromonas gingivalis (P. gingivalis) is one of the main periodontal bacteria. This pathogen was reported to enhance monocyte migration and adhesion to endothelial cells in atherosclerosis. The scavenger receptor lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) plays a pivotal role in atherogenesis. The aim of this study was to investigate whether LOX-1 modulates P. gingivalis-mediated monocyte migration and adhesion to endothelial cells and how it works. The results showed that the migration and adhesion of monocytic THP-1 cells to human umbilical vein endothelial cells (HUVECs) were significantly enhanced when HUVECs or THP-1 cells were challenged with P. gingivalis. Meanwhile, the expression level of LOX-1 in both HUVECs and THP-1 cells were also significantly increased by P. gingivalis stimulation. It is well known that ligand/receptor pairs monocyte chemoattractant protein-1 (MCP-1)/CC chemokine receptor 2 (CCR2), selectins/Integrins, and cell adhesion molecules (CAMs)/Integrins mediate monocyte migration and adhesion to endothelial cells. In this study, LOX-1 was demonstrated to be crucially involved in P. gingivalis-induced THP-1 cell migration and adhesion to HUVECs, by regulating expression of ligands MCP-1, intercellular adhesion molecule-1 (ICAM-1) and E-selectin in HUVECs and that of their receptors CCR2 and Integrin αMβ2 in THP-1 cells. The nuclear factor-kappa B (NF-κB) signaling pathway was proved to be involved in this process. In conclusion, LOX-1 plays a crucial role in P. gingivalis-induced monocyte migration and adhesion to endothelial cells. This result implies LOX-1 may act as a bridge in linking periodontitis to atherosclerosis.

Oxytocin inhibits ox-LDL-induced adhesion of monocytic THP-1 cells to human brain microvascular endothelial cells

The attachment of monocytes to human brain microvascular endothelial cells (HBMVEs) caused by oxidized low-density lipoprotein (ox-LDL) is associated with an early event and the pathological progression of cerebrovascular diseases. Oxytocin (OT) is a human peptide hormone that is traditionally used as a medication to facilitate childbirth. However, little information is available regarding the physiological function of OT in brain endothelial dysfunction. In the present study, our results indicate that the oxytocin receptor (OTR) was expressed in human brain microvascular endothelial cells (HBMVEs) and was upregulated in response to ox-LDL in a concentration-dependent manner. Notably, OT significantly suppressed ox-LDL-induced attachment of THP-1 monocytes to HBMVEs. Furthermore, we found that OT reduced the expression of adhesion molecules, such as VCAM-1 and E-selectin. Interestingly, it was shown that OT could restore ox-LDL-induced reduction of KLF4 in HBMVEs. Importantly, knockdown of KLF4 abolished the inhibitory effects of OT on ox-LDL-induced expressions of VCAM-1 and E-selectin as well as the adhesion of human monocytic THP-1 cells to endothelial HBMVEs. Mechanistically, we found that the stimulatory effects of OT on KLF4 expression are mediated by the MEK5/MEF2A pathway.

N-butanol extracts of Morinda citrifolia suppress advanced glycation end products (AGE)-induced inflammatory reactions in endothelial cells through its anti-oxidative properties

BackgroundAdvanced glycation end products (AGEs), senescent macroprotein derivatives formed during a normal aging process and acceleratedly under diabetic conditions, play a role in atherosclerotic cardiovascular disease. AGEs cause endothelial cell (EC) damage, an initial trigger for atherosclerosis through the interaction with a receptor for AGEs (RAGE). We have previously shown that n-butanol extracts of Morinda citrifolia (noni), a plant belonging to the family Rubiaceae, block the binding of AGEs to RAGE in vitro. In this study, we examined the effects of n-butanol extracts of noni on reactive oxygen species (ROS) generation and inflammatory reactions on AGE-exposed human umbilical vein ECs (HUVECs).MethodsHUVECs were treated with 100 μg/ml AGE-bovine serum albumin (AGE-BSA) or non-glycated BSA in the presence or absence of 670 ng/ml n-butanol extracts of noni for 4 h. Then ROS generation and inflammatory and gene expression in HUVECs were evaluated by dihydroethidium staining and real-time reverse transcription-polymerase chain reaction analyses, respectively. THP-1 cell adhesion to HUVECs was measured after 2-day incubation of AGE-BSA or BSA in the presence or absence of 670 ng/ml n-butanol extracts of noni.ResultsN-butanol extracts of noni at 670 ng/ml significantly inhibited the AGE-induced ROS generation and RAGE, intercellular adhesion molecule-1 and plasminogen activator inhibitor-1 gene expressions in HUVECs. AGEs significantly increased monocytic THP-1 cell adhesion to HUVECs, which was also prevented by 670 ng/ml n-butanol extracts of noni.ConclusionsThe present study demonstrated for the first time that N-butanol extracts of noni could suppress the AGE-induced inflammatory reactions in HUVECs through its anti-oxidative properties via blocking of the interaction of AGEs with RAGE. Inhibition of the AGE-RAGE axis by n-butanol extracts of noni may be a novel nutraceutical strategy for the treatment of cardiovascular disease.

Shear Stress Counteracts Endothelial CX3CL1 Induction and Monocytic Cell Adhesion.

Flow conditions critically regulate endothelial cell functions in the vasculature. Reduced shear stress resulting from disturbed blood flow can drive the development of vascular inflammatory lesions. On endothelial cells, the transmembrane chemokine CX3CL1/fractalkine promotes vascular inflammation by functioning as a surface-expressed adhesion molecule and by becoming released as soluble chemoattractant for monocytic cells expressing the receptor CX3CR1. Here, we report that endothelial cells from human artery, vein, or microvasculature constitutively express CX3CL1 when cultured under static conditions. Stimulation with TNFα under static or very low shear stress conditions strongly upregulates CX3CL1 expression. By contrast, CX3CL1 induction is profoundly reduced when cells are exposed to higher shear stress. When endothelial cells were grown and subsequently stimulated with TNFα under low shear stress, strong adhesion of monocytic THP-1 cells to endothelial cells was observed. This adhesion was in part mediated by transmembrane CX3CL1 as demonstrated with a neutralizing antibody. By contrast, no CX3CL1-dependent adhesion to stimulated endothelium was observed at high shear stress. Thus, during early stages of vascular inflammation, low shear stress typically seen at atherosclerosis-prone regions promotes the induction of endothelial CX3CL1 and monocytic cell recruitment, whereas physiological shear stress counteracts this inflammatory activation of endothelial cells.

PM2.5-induced oxidative stress increases adhesion molecules expression in human endothelial cells through the ERK/AKT/NF-κB-dependent pathway.

The aim of this study was to explore the intracellular mechanisms underlying the cardiovascular toxicity of air particulate matter (PM) with an aerodynamic diameter of less than 2.5 µm (PM2.5) in a human umbilical vein cell line, EA.hy926. We found that PM2.5 exposure triggered reactive oxygen species (ROS) generation, resulting in a significant decrease in cell viability. Data from Western blots showed that PM2.5 induced phosphorylation of Jun N-terminal kinase (JNK), extracellular signal regulatory kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and protein kinase B (AKT), and activation of nuclear factor kappa B (NF-κB). We further observed a significant increase in expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) in a time- and dose-dependent manner. Moreover, the adhesion of monocytic THP-1 cells to EA.hy926 cells was greatly enhanced in the presence of PM2.5 . However, N-acetylcysteine (NAC), a scavenger of ROS, prevented the increase of ROS generation, attenuated the phosphorylation of the above kinases, and decreased the NF-κB activation as well as the expression of ICAM-1 and VCAM-1. Furthermore, ERK inhibitor (U0126), AKT inhibitor (LY294002) and NF-κB inhibitor (BAY11-7082) significantly down-regulated PM2.5 -induced ICAM-1 and VCAM-1 expression as well as adhesion of THP-1 cells, but not JNK inhibitor (SP600125) and p38 MAPK inhibitor (SB203580), indicating that ERK/AKT/NF-κB is involved in the signaling pathway that leads to PM2.5 -induced ICAM-1 and VCAM-1 expression. These findings suggest PM2.5 -induced ROS may function as signaling molecules triggering ICAM-1 and VCAM-1 expressions through activating the ERK/AKT/NF-κB-dependent pathway, and further promoting monocyte adhesion to endothelial cells.

Adenosine Regulates the Proinflammatory Signaling Function of Thrombin in Endothelial Cells

The plasma level of the regulatory metabolite adenosine increases during the activation of coagulation and inflammation. Here we investigated the effect of adenosine on modulation of thrombin-mediated proinflammatory responses in HUVECs. We found that adenosine inhibits the barrier-disruptive effect of thrombin in HUVECs by a concentration-dependent manner. Analysis of cell surface expression of adenosine receptors revealed that A2A and A2B are expressed at the highest level among the four receptor subtypes (A2B > A2A > A1 > A3 ) on HUVECs. The barrier-protective effect of adenosine in response to thrombin was recapitulated by the A2A specific agonist, CGS 21680, and abrogated both by the siRNA knockdown of the A2A receptor and by the A2A -specific antagonists, ZM-241385 and SCH-58261. The thrombin-induced RhoA activation and its membrane translocation were both inhibited by adenosine in a cAMP-dependent manner, providing a molecular mechanism through which adenosine exerts a barrier-protective function. Adenosine also inhibited thrombin-mediated activation of NF-κB and decreased adhesion of monocytic THP-1 cells to stimulated HUVECs via down-regulation of expression of cell surface adhesion molecules, VCAM-1, ICAM-1, and E-selectin. Moreover, adenosine inhibited thrombin-induced elevated expression of proinflammatory cytokines, IL-6 and HMGB-1; and chemokines, MCP-1, CXCL-1, and CXCL-3. Taken together, these results suggest that adenosine may inhibit thrombin-mediated proinflammatory signaling responses, thereby protecting the endothelium from injury during activation of coagulation and inflammation.

Synergistic Effect of Histamine and TNF-α on Monocyte Adhesion to Vascular Endothelial Cells

The histamine level is high during allergic attacks, and patients with allergy may have chronic inflammatory conditions at which tumor necrosis factor (TNF)-α is extensively released by macrophages. Here, in vitro static and microfluidic flow assays were conducted to investigate the combined influence of histamine and TNF-α on adhesion of monocytic THP-1 cells to human umbilical vein endothelial cells (HUVEC). In a static assay, histamine stimulation of TNF-α-activated HUVEC elevated the number of attached THP-1 cells. In a flow assay, the number of crawling and firmly adherent THP-1 cells was higher on TNF-α + histamine activated HUVEC than on HUVEC activated by TNF-α alone. This synergistic effect of histamine and TNF-α is caused by the increased endothelial surface expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. Since the exposure of TNF-α-activated endothelium to histamine favors monocyte recruitment, it may be a serious risk factor for atherosclerosis and other inflammatory disorders.

89 NOVEL VASOPROTECTIVE ACTIONS OF NITROXYL (HNO) IN THE VASCULATURE

Background: Nitric oxide (NO•) plays a fundamental role as a vascular protectant with its well reported ability to induce vasorelaxation in human vessels and reduce leukocyte to endothelium adhesion, an initiating event of vascular inflammation. Nitroxyl (HNO), the one-electron reduced redox sibling of NO•, has distinct pharmacology to NO• and the effects of HNO in this context are not known. Objective: We aimed to determine the vasodilatory and anti-inflammatory actions of HNO using the HNO donor, Angeli's Salt (AS) compared to the NO• donor, GTN. Methods and Results: Cumulative concentration-response curves (CRCs) to AS or GTN were constructed in radial arteries (RA) and saphenous veins (SV) obtained from patients undergoing coronary artery graft surgery mounted in organ baths. AS induced vasorelaxation of RA (EC50 = 6.5 ± 0.1 M; n = 8) and SV (EC50 = 6.6 ± 0.1 M; n = 9) with similar potency to GTN, and AS-induced vasorelaxation was inhibited by the HNO scavenger L-cysteine (3 mM) but not the NO• scavenger hydroxocobalamin (100 μM). Adhesion of monocytic THP-1 cells to activated HUVECs (with tumour necrosis factor α (TNFα)) was decreased in a concentration-dependent manner by AS and GTN (P<0.05). Furthermore, experiments which allow the visualization of leukocyte-endothelium adhesion in isolated mouse (C57BL/6) aorta, using whole blood, showed that AS (10 μM) and GTN (10 μM) significantly attenuated (5 ± 2 and 3 ± 1 cells/field, respectively, n = 4-5;P<0.05) the TNFα-stimulated increase (16 ± 3 cells/field, n = 5-11) in leukocyte adhesion. Conclusions: This study shows for the first time that HNO can relax human blood vessels and reduce leukocyte-endothelium adhesion which will aid in determining the therapeutic potential of HNO donors.

αVβ3-integrin expression through ERK activation mediates cell attachment and is necessary for production of tumor necrosis factor alpha in monocytic THP-1 cells stimulated by phorbol myristate acetate

Macrophages play a key role in inflammation. Activated macrophages express adhesion molecules and produce tumor necrosis factor alpha (TNFα). Integrins are the main adhesion molecules that mediate binding to the extracellular matrix and they are involved in intracellular pathways. In the present study, human monocytic THP-1 cell adhesion to uncoated plastic plate was examined to investigate the regulatory mechanism of TNFα secretion. Addition of phorbol myristate acetate (PMA) for THP-1 cell activation induced cell adhesion in parallel with TNFα production. Among the mitogen-activated protein kinase pathways, the protein kinase C (PKC)–extracellular signal-regulated kinase (ERK) pathway was involved in αVβ3-integrin expression and PMA-induced cell adhesion. Flow cytometry and reverse transcription – quantitative polymerase chain reaction analysis revealed increased expression of matrix-binding integrins including integrin-αVβ3. Blockade of αVβ3-integrin by a specific antibody suppressed cell adhesion and TNFα production. These findings indicate that TNFα production from THP-1 cells is PKC–ERK, αVβ3-integrin and adhesion-dependent and its related pathway could be a target for TNFα-related diseases.

Kallistatin Inhibits Vascular Inflammation by Antagonizing Tumor Necrosis Factor-α–Induced Nuclear Factor κB Activation

Kallistatin is a plasma protein with anti-inflammatory properties. In this study, we investigated the role and mechanisms of kallistatin in inhibiting endothelial inflammation through its heparin-binding domain. We showed that recombinant wild-type kallistatin dose-dependently competed with tumor necrosis factor (TNF)-alpha binding to TNF-alpha receptor in endothelial cells, whereas kallistatin mutant at the heparin-binding domain had no effect. Kallistatin, but not kallistatin mutant at the heparin-binding domain, abrogated TNF-alpha-induced endothelial cell activation, as evidenced by inhibition of TNF receptor 1-associated death domain protein activation, inhibitor of nuclear factor kappaB-alpha degradation, nuclear factor kappaB translocation, and p38 mitogen-activated protein kinase phosphorylation, as well as cell adhesion molecule and cytokine expression. Moreover, kallistatin, but not kallistatin mutant at the heparin-binding domain, inhibited TNF-alpha-induced human monocytic THP-1 cell adhesion to endothelial cells and prevented vascular endothelial growth factor-induced endothelial permeability. In mice, kallistatin gene delivery prevented vascular leakage provoked by complement factor C5a, whereas delivery of kallistatin heparin mutant gene had no effect. Similarly, gene transfer of kallistatin, but not the kallistatin heparin mutant, inhibited collagen/adjuvant-induced arthritis in rats. These results indicate that kallistatin's heparin-binding site plays an essential role in preventing TNF-alpha-mediated endothelial activation and reducing vascular endothelial growth factor-induced vascular permeability, resulting in attenuation of vascular inflammation in cultured endothelial cells and animal models. This study identifies a protective role of kallistatin in vascular injury, thereby implicating the therapeutic potential of kallistatin for vascular and inflammatory diseases.

Pine Bark Extract Enzogenol Attenuated Tumor Necrosis Factor-α-Induced Endothelial Cell Adhesion and Monocyte Transmigration

The transmigration and extravasation of leukocytes across the endothelium that lines the vessel wall occurs in distinct multisteps first comprising rolling of the leukocytes over the endothelial cells, resulting in a tightly controlled and very complex system of leukocyte trafficking and transmigration. Vascular endothelial cells are an important target of proinflammatory cytokines modulating many genes involved in cell adhesion, thrombosis, and inflammatory responses. This study examined whether enzogenol blunts transendothelial migration of monocytes through tumor necrosis factor (TNF)-alpha-activated human umbilical vein endothelial cells (HUVEC). HUVECs were incubated with 10 ng/mL TNF-alpha for 6 h in the absence and presence of 5-50 microg/mL enzogenol. Expression of protein and mRNA of adhesion molecules in HUVEC were measured with Western blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR) assay. Monocytic THP-1 cell adhesion and transmigration were examined by calcein AM-staining and matrix metalloproteinase-9 (MMP-9) activity measured by gelatin zymography. Intracellular localization of nuclear factor-kappa B (NF-kappaB) p65 revealed involvement of NF-kappaB signaling. TNF-alpha markedly induced protein expression of cell adhesion molecule and E-selectin with increasing mRNA levels in HUVEC. Nontoxic enzogenol at 5-25 microg/mL attenuated the expression of all adhesion molecules in a dose-dependent fashion. Consistently, enzogenol suppressed the enhanced THP-1 cell adhesion onto TNF-alpha-activated HUVEC through diminishing integrin beta2 induction. In TNF-alpha-activated HUVEC were observed IkappaB dissociation and NF-kappaB nuclear translocation, which was ameliorated by enzogenol. Furthermore, enzogenol hampered the transendothelial migration of THP-1 cells by increasing MMP-9 secretion and activity. Blunting induction of cell adhesion molecules by enzogenol was mediated by their interference with the NF-kappaB-dependent transcription pathways. Thus, enzogenol may have therapeutic potential targeting inflammatory response-associated atherosclerosis.

Potent anti-inflammatory effects of low-dose proteasome inhibition in the vascular system

Proteasome inhibitors are considered to have anti-inflammatory therapeutic potential. However, recent reports addressing proteasome inhibition in the vascular system are controversial, ranging from beneficial anti-inflammatory and anti-oxidative effects to potentiation of inflammation and oxidative stress. This study was based on the hypothesis that the divergent effects might be a result of a differential and dose-dependent responsiveness of vascular cells to proteasome inhibitors. We tested whether low doses of proteasome inhibitors would favor anti-inflammatory effects in vascular cells in vitro and in vivo. Human umbilical vein endothelial cells (HUVEC) were preincubated with proteasome inhibitors MG132 and MG262 at concentrations that did not affect cell viability during a 24-h treatment. Upon addition of tumor necrosis factor alpha (TNF-alpha) the induced expression of adhesion molecules and the adhesion of monocytic THP-1 cells to HUVECs was significantly lowered. However, nuclear translocation of NF-kappaB was only slightly diminished. Low-dose pretreatment with proteasome inhibitors decreased TNF-alpha-induced generation of reactive oxygen species in HUVEC. Bortezomib was administered at a dose of 50 microg/kg body weight to Dahl salt-sensitive rats (DSSR) on high-salt diet. This low-dose proteasome inhibition led to decreased hypertension-induced oxidative stress and reduced expression of vascular cell adhesion molecule 1 (VCAM-1) in the aortae.

Effect of serum concentration on adhesion of monocytic THP-1 cells onto cultured EC monolayer and EC-SMC co-culture

The adhesion of monocytes to the endothelium following accumulation of low-density lipoprotein (LDL) in subendothelial spaces is an important step in the development of intimal hyperplasia in arterially implanted vein grafts and atherosclerosis in both animals and humans. However, it is not well known how serum factors affect the adhesion of monocytes. We have studied the effect of fetal calf serum (FCS), which we considered a source of LDL, on the adhesion of monocytes to endothelial cells (ECs) by using human monocytic THP-1 cells and both a monolayer of cultured bovine aortic endothelial cells (EC monoculture) and a co-culture with bovine aortic smooth muscle cells (EC-SMC co-culture). It was found that the addition of FCS to the medium greatly affected the adhesion of THP-1 cells, and the higher the concentration of FCS in the medium, the greater the adhesion of THP-1 cells to endothelial cells. Adhesion of THP-1 cells to an EC-SMC co-culture was approximately twofold greater than that to an EC monoculture, and after adhering to endothelial cells, many THP-1 cells transmigrated into the layer of smooth muscle cells. The results suggest that the elevation of the LDL (cholesterol) level in blood provides a favorable condition for the development of intimal hyperplasia and atherosclerosis by promoting the adhesion of monocytes to the endothelium and their subsequent migration into subendothelial spaces.

Cardiotrophin-1 stimulates intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 in human aortic endothelial cells

Intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) play critical roles in mediating monocyte adhesion to the vascular endothelium and monocyte migration into the subendothelial regions of the vessels. Inasmuch as cardiotrophin-1 (CT-1), an IL-6-type cytokine, was expressed in human atherosclerotic plaque, we examined whether CT-1 induces monocyte adhesion and migration by stimulating gene and protein expressions of ICAM-1 and MCP-1 in human aortic endothelial cells (HAECs). Immunocytochemistry revealed that CT-1 increased intensity of ICAM-1 and MCP-1 immunoreactivity in HAECs. Adhesion assay and chemotaxis assay revealed that CT-1 increased human monocytic THP-1 cell adhesion to HAECs and promoted chemotaxis in THP-1 cells, which were attenuated by anti-ICAM-1 and anti-MCP-1 antibody, respectively. Western blot analysis showed that CT-1 increased phosphorylation of ERK1/2 MAP kinase, p38 MAP kinase, and Akt and that their inhibitors, PD-98059, SB-203580, and LY-294002, respectively, inhibited phosphorylation. RNase protection assay and ELISA demonstrated that CT-1 increased gene and protein expressions of ICAM-1 and MCP-1. EMSA revealed that CT-1 enhanced NF-kappaB DNA-binding activity. CT-1-mediated upregulation of ICAM-1 and MCP-1 was suppressed by PD-98059, SB-203580, LY-294002, and parthenolide. The present study demonstrates that CT-1 promotes monocyte adhesion and migration by stimulating ICAM-1 and MCP-1 through mechanisms that involve ERK1/2 MAP kinase, p38 MAP kinase, phosphatidylinositol 3-kinase, and NF-kappaB pathways and suggests that CT-1 plays an important role in the pathophysiology of vascular inflammation and atherosclerosis.

Bone Morphogenetic Protein-2 Induces Proinflammatory Endothelial Phenotype

The transforming growth factor-beta superfamily member bone morphogenetic protein-2 (BMP-2) is up-regulated in atherosclerotic arteries; however, its effects on the endothelium are not well characterized. Using microdissected coronary arterial endothelial cells (CAECs) and cultured primary CAECs, we demonstrated endothelial mRNA expression of BMP-2 and BMP-4. The proinflammatory cytokine tumor necrosis factor-alpha and H2O2 significantly increased endothelial expression of BMP-2 but not BMP-4. In organ culture, BMP-2 substantially decreased relaxation of rat carotid arteries to acetylcholine and increased production of reactive oxygen species, events inhibited by pharmacologically blocking protein kinase C (PKC) or NAD(P)H oxidase. BMP-2 activated nuclear factor-kappaB in CAECs, and BMP-2 and BMP-4 substantially increased adhesion of monocytic THP-1 cells, which was reduced by pharmacologically inhibiting p42/44 MAP kinase pathway (also by siRNA down-regulating ERK-1/2) or PKC. Incubation of rat carotid arteries with BMP-2 ex vivo also increased adhesion of mononuclear cells to the endothelium, requiring p42/44 MAP kinase and PKC. Western blotting showed that in CAECs and carotid arteries BMP-2 elicited phosphorylation of p42/44 MAP kinase, which was reduced by blocking MAP kinase kinase and PKC. Collectively, expression of BMP-2 is regulated by proinflammatory stimuli, and increased levels of BMP-2 induce endothelial dysfunction, oxidative stress, and endothelial activation. Thus, the proinflammatory effects of BMP-2 may play a role in vascular pathophysiology.

ACE Inhibition Lowers Angiotensin-II-Induced Monocyte Adhesion to HUVEC by Reduction of p65 Translocation and AT1 Expression

Angiotensin-converting enzyme (ACE) inhibitors interfere with several key events of vascular inflammation resulting in impressive reductions in coronary vascular events. However, in human arteries ACE inhibitors block the production of angiotensin II (AngII) incompletely because of the involvement of alternative pathways in local AngII formation. Therefore, our study concentrated on the presumed modulation by ACE inhibition of local AngII-mediated inflammatory actions by a mechanism independent of blockage of AngII formation. We analyzed the effect of the ACE inhibitor ramiprilat on AngII-dependent cell adhesion molecule (CAM) expression and adhesion of monocytic THP-1 cells to endothelial cells. AngII induced upregulation of P-selectin, VCAM-1 and ICAM-1 on endothelial cells via activation of AT1, which was correlated with enhanced THP-1 adhesion in flow chamber assays. Both enhanced adhesion and adhesion molecule expression were significantly reduced by pretreatment with ramiprilat. Ramiprilat reduced AT1 expression on endothelial cells and decreased the AngII-induced p65 translocation into the nucleus. Diminished AT1 expression and adhesion molecule expression in response to ramiprilat treatment were partially reversed after incubation with a bradykinin 2 receptor antagonist, suggesting that elevated bradykinin levels under ACE inhibition may be involved in the beneficial effect of ACE inhibitors. Thus, modulation of the local AngII system by ramiprilat may at least in part contribute to the benefits of ACE inhibition in the treatment of atherosclerotic diseases.

Shear Stress Inhibits Smooth Muscle Cell–Induced Inflammatory Gene Expression in Endothelial Cells

Vascular endothelial cells (ECs) are influenced by shear stress and neighboring smooth muscle cells (SMCs). We investigated the inflammation-relevant gene expression in EC/SMC cocultures under static condition and in response to shear stress. Under static condition, DNA microarrays and reverse-transcription polymerase chain reaction identified 23 inflammation-relevant genes in ECs whose expression was significantly affected by coculture with SMCs, with 18 upregulated and 5 downregulated. Application of shear stress (12 dynes/cm2) to the EC side of the coculture for 6 hours inhibited most of the proinflammatory gene expressions in ECs induced by coculture with SMCs. Inhibition of nuclear factor-kappaB (NF-kappaB) activation by the p65-antisense, lactacystin, and N-acetyl-cysteine blocked the coculture-induced EC expression of proinflammatory genes, indicating that the NF-kappaB binding sites in the promoters of these genes play a significant role in their expression as a result of coculture with SMCs. Chromatin immunoprecipitation assays demonstrated the in vivo regulation of NF-kappaB recruitment to selected target promoters. Shear stress inhibited the SMC coculture-induced NF-kappaB activation in ECs and monocytic THP-1 cell adhesion to ECs. Our findings suggest that shear stress plays an inhibitory role in the proinflammatory gene expression in ECs located in close proximity to SMCs.