Monocyte-endothelial Cell Adhesion Research Articles

In vitro systems toxicology-based assessment of the potential modified risk tobacco product CHTP 1.2 for vascular inflammation- and cytotoxicity-associated mechanisms promoting adhesion of monocytic cells to human coronary arterial endothelial cells

Cigarette smoking causes cardiovascular diseases. Heating tobacco instead of burning it reduces the amount of toxic compounds in the aerosol and may exert a reduced impact on health compared with cigarette smoke. Aqueous extract from the aerosol of a potential modified risk tobacco product, the Carbon Heated Tobacco Product (CHTP) 1.2, was compared in vitro with aqueous extract from the smoke of a 3R4F reference cigarette for its impact on the adhesion of monocytic cells to artery endothelial cells. Human coronary artery endothelial cells (HCAEC) were treated for 4 h with conditioned media from human monocytic Mono Mac 6 (MM6) cells exposed to CHTP1.2 or 3R4F extracts for 2 h or directly with those extracts freshly generated. In vitro monocyte-endothelial cell adhesion was measured concomitantly with inflammatory, oxidative stress, cytotoxicity, and death markers. Furthermore, transcriptomics analyses enabled to quantify the level of perturbation in HCAECs, and provide biological interpretation for the underlying molecular changes following exposure to 3R4F or CHTP1.2 extract. Our systems toxicology study demonstrated that approximately 10–15-fold higher concentrations of the CHTP 1.2 aerosol extract were needed to elicit similar effects as the 3R4F smoke extract on cardiovascular disease-relevant inflammation and cytotoxicity-related mechanisms and markers investigated in vitro.

Adropin Contributes to Anti-Atherosclerosis by Suppressing Monocyte-Endothelial Cell Adhesion and Smooth Muscle Cell Proliferation.

Adropin, a peptide hormone expressed in liver and brain, is known to improve insulin resistance and endothelial dysfunction. Serum levels of adropin are negatively associated with the severity of coronary artery disease. However, it remains unknown whether adropin could modulate atherogenesis. We assessed the effects of adropin on inflammatory molecule expression and human THP1 monocyte adhesion in human umbilical vein endothelial cells (HUVECs), foam cell formation in THP1 monocyte-derived macrophages, and the migration and proliferation of human aortic smooth muscle cells (HASMCs) in vitro and atherogenesis in Apoe−/− mice in vivo. Adropin was expressed in THP1 monocytes, their derived macrophages, HASMCs, and HUVECs. Adropin suppressed tumor necrosis factor α-induced THP1 monocyte adhesion to HUVECs, which was associated with vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 downregulation in HUVECs. Adropin shifted the phenotype to anti-inflammatory M2 rather than pro-inflammatory M1 via peroxisome proliferator-activated receptor γ upregulation during monocyte differentiation into macrophages. Adropin had no significant effects on oxidized low-density lipoprotein-induced foam cell formation in macrophages. In HASMCs, adropin suppressed the migration and proliferation without inducing apoptosis via ERK1/2 and Bax downregulation and phosphoinositide 3-kinase/Akt/Bcl2 upregulation. Chronic administration of adropin to Apoe−/− mice attenuated the development of atherosclerotic lesions in the aorta, with reduced the intra-plaque monocyte/macrophage infiltration and smooth muscle cell content. Thus, adropin could serve as a novel therapeutic target in atherosclerosis and related diseases.

Curcumin Attenuates Expression of Adhesion Molecules by IL‐1 β in HUVECs

Atherosclerosis, a progressive pathological disorder inducing cardiovascular and cerebrovascular diseases, is a leading cause of human morbidity and mortality worldwide. The expression of cell adhesion molecules such as intercellular adhesion molecule (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), and adhesion molecule‐1 (E‐selectin) by the endothelium and the attachment of monocytes to endothelium may play a pivotal role in the early atherogenic process. Curcumin is a polyphenol obtained from the root of the turmeric (Curcuma longa), and is the main component of curry powder for giving a specific flavor and yellow color. Previous studies indicated that curcumin exhibits a variety of pharmacological effects including anti‐inflammatory, antitumor, antioxidant, anti‐hypertensive, antiviral, anti‐obesity, anti‐infectious activities and wound‐healing properties. In this study, efforts were made to identify curcumin may aid in the prevention of monocyte‐endothelial cell adhesion induced atherosclerosis. Our results indicated that curcumin could inhibit the expression of adhesion molecules, including ICAM‐1, VCAM‐1, and E‐selectin, and futher decrease the adhesiveness to a human monocytic cell line (U937) in human umbilical vein endothelial cells (HUVECs). These results demonstrated that curcumin has inhibitory effect on proanthersclerotic mechanism in vitro.Support or Funding InformationThis study was supported by NSC 100‐2313‐B‐309‐003 to A‐C Cheng and NSC 95‐2815‐C‐039‐019‐B to Y‐M Yu.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Porphyromonas gingivalis ATCC 33277 promotes intercellular adhesion molecule-1 expression in endothelial cells and monocyte-endothelial cell adhesion through macrophage migration inhibitory factor

BackgroundPorphyromonas gingivalis (P. gingivalis), one of the main pathogenic bacteria involved in periodontitis, induces the expression of intercellular adhesion molecule − 1 (ICAM-1) and monocyte-endothelial cell adhesion. This effect plays a pivotal role in atherosclerosis development. Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine and critically affects atherosclerosis pathogenesis. In this study, we tested the involvement of MIF in the P. gingivalis ATCC 33277-enhanced adhesive properties of endothelial cells.ResultsEndothelial MIF expression was enhanced by P. gingivalis ATCC 33277 infection. The MIF inhibitor ISO-1 inhibited ICAM-1 production in endothelial cells, and monocyte-endothelial cell adhesion was induced by P. gingivalis ATCC 33277 infection. However, the addition of exogenous human recombinant MIF to P. gingivalis ATCC 33277-infected endothelial cells facilitated monocyte recruitment by promoting ICAM-1 expression in endothelial cells.ConclusionsThese experiments revealed that MIF in endothelial cells participates in the pro-atherosclerotic lesion formation caused by P. gingivalis ATCC 33277 infection. Our novel findings identify a more detailed pathological role of P. gingivalis ATCC 33277 in atherosclerosis.

A major daidzin metabolite 7,8,4′-trihydroxyisoflavone found in the plasma of soybean extract-fed rats attenuates monocyte-endothelial cell adhesion

Among many functional foods and their phytochemicals, ingestion of soybean (Glycine max) is highly correlated to reduced risk of cardiovascular diseases. Validation of potential health benefits of functional foods requires information about the bioavailability and metabolism of bioactive compounds. In this context, several phase I and II metabolites of isoflavones were target-analyzed in the plasma of rats acutely supplemented with soybean embryo extract. A daidzein metabolite, 7,8,4′-trihydroxyisoflavone (7,8,4′-THI), was found to have the highest average area under curve value (574.3±112.8). Therefore, its potential prevention effect on atherosclerosis was investigated using monocyte-endothelial cell adhesion assay. Different from its precursor daidzein or daidzin, 7,8,4′-THI attenuated adhesion of THP-1 monocytes to tumor necrosis factor-alpha (TNF-α) stimulated human umbilical vein endothelial cells (HUVECs). In addition, 7,8,4′-THI significantly downregulated TNF-α stimulated the expression of vascular cell adhesion molecule-1 and monocyte chemotactic protein-1 and phosphorylation of IκB kinase and IκBα involved in the initiation of atherosclerosis in HUVECs. Therefore, 7,8,4′-THI, a highly bioavailable hydroxylated isoflavone metabolite, has potential anti-atherosclerotic effect via inhibiting monocyte-endothelial adhesion.

5-(3′,4′-Dihydroxyphenyl-γ-valerolactone), a Major Microbial Metabolite of Proanthocyanidin, Attenuates THP-1 Monocyte-Endothelial Adhesion

Several metabolomics of polymeric flavan-3-ols have reported that proanthocyanidins are extensively metabolized by gut microbiota. 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone (DHPV) has been reported to be the major microbial metabolite of proanthocyanidins. We demonstrated that DHPV has stronger prevention effect on tumor necrosis factor (TNF)-α-stimulated adhesion of THP-1 human monocytic cells to human umbilical vein endothelial cells compared to its potential precursors such as procyanidin A1, A2, B1 and B2, (+)catechin, (−)epicatechin and its microbial metabolites such as 3-(3,4-dihydroxyphenyl)propionic acid and 2-(3,4-dihydroxyphenyl)acetic acid. Mechanism study showed that DHPV prevents THP-1 monocyte-endothelial cell adhesion by downregulating TNF-α-stimulated expressions of the two biomarkers of atherosclerosis such as vascular cell adhesion molecule-1 and monocyte chemotactic protein-1, activation of nuclear factor kappa B transcription and phosphorylation of I kappa-B kinase and IκBα. We suggested that DHPV has higher potentiality in prevention of atherosclerosis among the proanthocyanidin metabolites.

Ligands of toll-like receptors 2/4 differentially alter markers of inflammation, adhesion and angiogenesis by monocytes from women with pre-eclampsia in co-culture with endothelial cells

Pre-eclampsia (PE) is characterized by an exaggerated systemic inflammatory response and generalized endothelial dysfunction. We have recently demonstrated that fibrinogen, an endogenous ligand of Toll-like receptor (TLR) 4, activates monocytes from women with pre-eclampsia (Al-ofi et al., 2014). Using an experimental co-culture model of primary human monocytes (derived from 9 women with PE (GA=33.18±5.8) and 9 normotensive pregnant women, NP (GA=33.15±4.0)) and human umbilical venous endothelial cells (HUVECs), we compared the effects of fibrinogen and lipopolysaccharide (LPS, bacterial ligand to TLR4) on the expression levels of inflammatory cytokines (IL-6 and IL-1β), chemokines (IL-8 and MCP-1), and anti-angiogenic factor (soluble fms-like tyrosine kinase-1,sFLT-1), as well as the soluble vascular cell adhesion molecule-1 (sVCAM-1). Cytokines, VEGF and sVCAM-1 were measured in the supernatant media by cytometric array. The levels of sFLT-1 were measured by ELISA. Fibrinogen induced greater expression levels of IL-1β and VCAM-1 from PE HUVEC-monocyte co-culture than from NP HUVEC-monocyte co-culture (P<0.05), similar to the effects of LPS. In contrast, unlike LPS, fibrinogen suppressed IL-6, IL-8, MCP-1 and sFLT-1 production by co-cultures that included PE monocytes compared to those with NP monocytes (P<0.05). In conclusion, fibrinogen promotes monocyte-endothelial cell adhesion and angiogenesis and suppresses the expression of some inflammatory markers in pre-eclampsia. Although the physiological implications of these intriguing observations are unclear our findings suggest that fibrinogen contributes to the regulation of cell adhesion, angiogenesis and inflammation by mechanisms not wholly dependent on TLR4 stimulation.

STING-IRF3 Triggers Endothelial Inflammation in Response to Free Fatty Acid-Induced Mitochondrial Damage in Diet-Induced Obesity.

Metabolic stress in obesity induces endothelial inflammation and activation, which initiates adipose tissue inflammation, insulin resistance, and cardiovascular diseases. However, the mechanisms underlying endothelial inflammation induction are not completely understood. Stimulator of interferon genes (STING) is an important molecule in immunity and inflammation. In the present study, we sought to determine the role of STING in palmitic acid-induced endothelial activation/inflammation. In cultured endothelial cells, palmitic acid treatment activated STING, as indicated by its perinuclear translocation and binding to interferon regulatory factor 3 (IRF3), leading to IRF3 phosphorylation and nuclear translocation. The activated IRF3 bound to the promoter of ICAM-1 (intercellular adhesion molecule 1) and induced ICAM-1 expression and monocyte-endothelial cell adhesion. When analyzing the upstream signaling, we found that palmitic acid activated STING by inducing mitochondrial damage. Palmitic acid treatment caused mitochondrial damage and leakage of mitochondrial DNA into the cytosol. Through the cytosolic DNA sensor cGAS (cyclic GMP-AMP synthase), the mitochondrial damage and leaked cytosolic mitochondrial DNA activated the STING-IRF3 pathway and increased ICAM-1 expression. In mice with diet-induced obesity, the STING-IRF3 pathway was activated in adipose tissue. However, STING deficiency (Stinggt/gt ) partially prevented diet-induced adipose tissue inflammation, obesity, insulin resistance, and glucose intolerance. The mitochondrial damage-cGAS-STING-IRF3 pathway is critically involved in metabolic stress-induced endothelial inflammation. STING may be a potential therapeutic target for preventing cardiovascular diseases and insulin resistance in obese individuals.

TNF-\u03b1 regulates the proteolytic degradation of ST6Gal-1 and endothelial cell-cell junctions through upregulating expression of BACE1

Endothelial dysfunction and monocyte adhesion to vascular endothelial cells are two critical steps in atherosclerosis development, and emerging evidence suggests that protein sialylation is involved in these processes. However, the mechanism underlying this phenomenon remains incompletely elucidated. In this study, we demonstrated that treatment with the proinflammatory cytokine TNF-α disrupted vascular endothelial cell-cell tight junctions and promoted monocyte endothelial cell adhesion. Western blotting and Sambucus nigra lectin (SNA) blotting analyses revealed that TNF-α treatment decreased α-2, 6-sialic acid transferase 1 (ST6Gal-I) levels and downregulated VE-Cadherin α-2, 6 sialylation. Further analysis demonstrated that TNF-α treatment upregulated β-site amyloid precursor protein enzyme 1 (BACE1) expression, thus resulting in sequential ST6Gal-I proteolytic degradation. Furthermore, our results revealed that PKC signaling cascades were involved in TNF-α-induced BACE1 upregulation. Together, these results indicated that the proinflammatory cytokine TNF-α impairs endothelial tight junctions and promotes monocyte-endothelial cell adhesion by upregulating BACE1 expression through activating PKC signaling and sequentially cleaving ST6Gal-I. Thus, inhibition of BACE1 expression may be a new approach for treating atherosclerosis.

29 Fibrinogen alters the expression of markers of inflammation, adhesion and angiogenesis by monocytes from women with pre-eclampsia in co-culture with vascular endothelial cells

Objectives Pre-eclampsia (PE) is characterized by an exaggerated systemic inflammatory response and generalized endothelial dysfunction. Having recently demonstrated that fibrinogen, an endogenous ligand of TLR4, activates monocytes from women with pre-eclampsia (Al-ofi et al., 2014), we sought to determine whether fibrinogen alters the expression of markers of inflammation, cell adhesion and angiogenesis by monocytes from women with pre-eclampsia, in co-culture with human umbilical venous endothelial cells (HUVEC), in a manner similar to the exogenous TLR ligand LPS, compared to normotensive women. Methods HUVECs were isolated from umbilical cords, cultured, passaged and seeded onto gelatin-coated 12-well tissue culture plates until they reached 80–90% of confluence. Then monocytes, isolated from 9 PE (GA=33.6±3.0) and 9 normal pregnant (GA=31.6±3.8) women, were plated on top of HUVECs at the fractional rate of 1 monocyte to 5 HUVECs. Mono- and co-cultures were stimulated with LPS and fibrinogen. Flow cytometry was used to confirm monocytes and endothelial cells in the model. We determined the expression levels of inflammatory cytokines (IL-6 and IL-1 β ), chemokines (IL-8 and MCP-1), angiogenic (VEGF) and anti-angiogenic (sFLT-1) factors, as well as adhesion molecule (sVCAM-1) in supernatant medium by cytometric array. ELISA was used to measure sFLT-1. Results Fibrinogen induced greater amounts of IL-1 β and VCAM-1 from PE co-culture than from NP co- culture ( P P Conclusion Fibrinogen promotes monocyte-endothelial cell adhesion and angiogenesis but suppresses the expression of inflammatory markers in pre-eclampsia. Although the physiological implications of these intriguing observations are unclear our findings suggest that fibrinogen contributes to the regulation of cell adhesion, angiogenesis and inflammation by mechanisms not wholly dependent on TLR4 stimulation.

Tm7sf2 may participate in the healing of burn wounds.

The transmembrane7 superfamily member2 (Tm7sf2) gene been reported to be involved in the reduced cholesterol levels of patients with large burn areas. To investigate the importance of Tm7sf2 in the burn wound healing process, a total of 10Sprague‑Dawley rats underwent electrical burns. Blood serum was collected for the culture of HaCaT human keratinocyte cells. Tm7sf2 small interfering RNAs (siRNAs) were prepared and transfected into the normal‑ and burn serum‑cultured HaCaT cells. Monocyte‑endothelial cellular adhesion ability and cell proliferation, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the levels of autophagy proteins Beclin1 and LC3‑II, which were detected by western blot assay and real time‑quantitative polymerase chain reaction, and it was identified that Tm7sf2 siRNAs were successfully transfected. Cell proliferation was reduced and monocyte-endothelial cell adhesion increased in the burn serum group compared with the normal serum group (P<0.05). Additionally, the expression levels of Beclin1 and LC3‑II were increased, and the addition of siTm7sf2 had a similar effect as the burn serum. Thus, Tm7sf2 may take part in the burn wound healing process by interacting with LC3‑II and Beclin1, and targeting Tm7sf2 may have therapeutic benefits.

Ulmus davidiana ethanol extract inhibits monocyte adhesion to tumor necrosis factor-alpha-stimulated endothelial cells

BackgroundUlmus davidiana var. japonica Rehder (UD) has long been used in traditional folk medicine in Asia. This study is designed to investigate the antiadhesive activity of the ethanol extract of UD (UDE) and its underlying mechanisms in cultured endothelial cells. MethodsThe dried root bark of UD was extracted with 80% (v/v) ethanol. The antiadhesive activity of the UDE was investigated in cultured human umbilical vein endothelial cells and human embryonic kidney epithelial 293T (HEK 293T) cells stably transfected with pGL3-vascular cell adhesion molecule (VCAM)-1-luc. Monocyte adhesion in endothelial cells was induced by tumor necrosis factor-alpha (TNF-α), and the protective effects of UDE on monocyte–endothelial cell adhesion, VCAM-1 expression, reactive oxygen species production, and nuclear factor-κB activity were determined. ResultsExposure to UDE at a concentration of 3–30μg/mL for 24hours produced no detectable cytotoxicity in human umbilical vein endothelial cells, but it significantly inhibited TNF-α-induced monocyte adhesion and VCAM-1 expression. TNF-α treatment of HEK 293T/VCAM-1-luc cells resulted in increased luciferase activity of the VCAM-1 promoter, which was inhibited by treatment with UDE. Additionally, TNF-α-induced reactive oxygen species generation, nuclear translocation of nuclear factor-κB, and IκBα degradation in human umbilical vein endothelial cells were effectively reduced by treatment with 30μg/mL of UDE. ConclusionOur results indicated that UDE treatment inhibited TNF-α-induced monocyte adhesion in endothelial cells, suggesting that UD may reduce vascular endothelial inflammation.

A novel anti-inflammatory mechanism of high density lipoprotein through up-regulating annexin A1 in vascular endothelial cells

High density lipoprotein (HDL) as well as annexin A1 have been reported to be associated with cardiovascular protection. However, the correlation between HDL and annexin A1 was still unknown. In this study, HDL increased endothelial annexin A1 and prevented the decrease of annexin A1 in TNF-α-activated endothelial cells in vitro and in vivo, and above effects were attenuated after knockdown of annexin A1. Annexin A1 modulation affected HDL-mediated inhibition of monocyte adhesion to TNF-α-activated endothelium (45.2±13.7% decrease for annexin A1 RNA interference; 78.7±16.3% decrease for anti-Annexin A1 antibody blocking; 11.2±6.9% increase for Ad-ANXA1 transfection). Additionally, HDL up-regulated annexin A1 through scavenger receptor class B type I, involving ERK, p38MAPK, Akt and PKC signaling pathways, and respective inhibitors of these pathways attenuated HDL-induced annexin A1 expression as well as impaired HDL-mediated inhibition of monocyte–endothelial cell adhesion. Apolipoprotein AI also increased annexin A1 and activated similar signaling pathways. Endothelial annexin A1 from apolipoprotein AI knockout mice was decreased in comparison to that from wild type mice. Finally, HDL-induced annexin A1 inhibited cell surface VCAM-1, ICAM-1 and E-selectin, and secretion of MCP-1, IL-8, VCAM-1 and E-selectin, thereby inhibiting monocyte adhesion.

Abstract 13138: Antioxidant-1, a Copper Transport Protein, promotes Inflammatory Neovascularization via Chaperone and Transcription Factor Function

Copper (Cu), an essential micronutrient, has been implicated in inflammation and angiogenesis with unknown mechanism. Cu transport protein, antioxidant-1 (Atox1) functions not only as Cu chaperone to activate secretory Cu enzymes (including lysyl oxidase (LOX)) via Cu transporter ATP7A, but also as Cu-dependent transcription factor. However, role of Atox1 in inflammatory neovascularization has not been investigated. Here we show that Atox1 expression is markedly upregulated in patients and mice with critical limb ischemia. Together with immunohistochemistry, micro-CT analysis and sponge implant assay, Atox1 KO mice exhibit impaired ischemia induced perfusion recovery, arteriogenesis/angiogenesis and inflammatory cell infiltration (46%). Furthermore, bone marrow (BM) reconstitution and adenovirus mediated Atox1 gene transfer in Atox1 KO mice show that Atox1 in tissue resident endothelial cells (ECs), but not in BM cells, is essential for neovascularization and recruitment of inflammatory cells which release angiogenic cytokines TNFα and VEGF. Mechanistically, Atox1 depletion of mice or ECs shows inhibition of ischemia (3.4-fold) and VEGF (1.6 fold) induced extracellular matrix/secretory Cu enzyme LOX activity in Cu/ATP7A-dependent manner, resulting in impaired (42%) capillary tube formation. Moreover, Atox1 knockdown in ECs inhibits ROS production (2.5 fold) and its downstream NFκB-DNA binding, VCAM1/ICAM1 expression and monocyte adhesion in ECs activated by TNFα, in Cu-dependent and ATP7A-independent manner, which are further supported by in vivo DHE staining and Bioluminescence imaging of NFκB reporter mice. Of note, Atox1 translocates to the nucleus (90%) in response to TNFα, thereby increasing transcription of p47phox NADPH oxidase by binding to its promoter in Cu-dependent manner. p47phox re-expression rescued reduced TNFα -induced VCAM1 expression in Atox1 KO ECs. In summary, Atox1 plays an important role in inflammatory neovascularization via Cu chaperone for LOX to increase angiogenesis as well as transcription factor for p47phox to promote ROS and inflammatory responses. Thus, Atox1 is a potential therapeutic target for promoting tissue repair in ischemic cardiovascular disease.

Copper Transport Protein Antioxidant-1 Promotes Inflammatory Neovascularization via Chaperone and Transcription Factor Function

Copper (Cu), an essential micronutrient, plays a fundamental role in inflammation and angiogenesis; however, its precise mechanism remains undefined. Here we uncover a novel role of Cu transport protein Antioxidant-1 (Atox1), which is originally appreciated as a Cu chaperone and recently discovered as a Cu-dependent transcription factor, in inflammatory neovascularization. Atox1 expression is upregulated in patients and mice with critical limb ischemia. Atox1-deficient mice show impaired limb perfusion recovery with reduced arteriogenesis, angiogenesis, and recruitment of inflammatory cells. In vivo intravital microscopy, bone marrow reconstitution, and Atox1 gene transfer in Atox1−/− mice show that Atox1 in endothelial cells (ECs) is essential for neovascularization and recruitment of inflammatory cells which release VEGF and TNFα. Mechanistically, Atox1-depleted ECs demonstrate that Cu chaperone function of Atox1 mediated through Cu transporter ATP7A is required for VEGF-induced angiogenesis via activation of Cu enzyme lysyl oxidase. Moreover, Atox1 functions as a Cu-dependent transcription factor for NADPH oxidase organizer p47phox, thereby increasing ROS-NFκB-VCAM-1/ICAM-1 expression and monocyte adhesion in ECs inflamed with TNFα in an ATP7A-independent manner. These findings demonstrate a novel linkage between Atox1 and NADPH oxidase involved in inflammatory neovascularization and suggest Atox1 as a potential therapeutic target for treatment of ischemic disease.

The Role and Mechanism of VCAM-1 in 2-Methoxyestradiol Inhibiting Monocyte-Endothelial Cell Adhesions

2-Methoxyestradiol (2-ME) is a major endogenous metabolite of estrogen, which formed by catalysis of cytochrome P450 (CYP450) and catechins-O-methoxy transferase (COMT), has an important role in atherosclerosis (AS), but the underlying mechanisms remain obscure. In our work, we investigated the role and mechanism of 2-ME inhibiting monocyte-endothelial cell adhesion. In vitro studies using monocyte-endothelial cell adhesion assay and cell immunofluorescence method showed that lipopolysaccharide (LPS) promoted monocyte-endothelial cell adhesion, these effects were blocked by 2-ME through a dose- and time-dependent manner. Furthermore, Vascular cell adhesion molecular-1 (VCAM-1) specific antibody (anti- VCAM-1, 1:1000) were pre-incubated the cells for 48 h, and by monocyte-endothelial cell adhesion experiment, results showed that anti-VCAM-1 can significantly inhibit the LPS-induced monocyte-endothelial cell adhesion, indicating VCAM-1 may be the key molecule of the LPS-nduced cell adhesion. Moreover, through Western Blot and monocyte-endothelial cell adhesion assay observing, results showed that 2-ME (10^(-5) M) pretreatment for 48 h was strongest inhibited the expression of VCAM-1, NF-_κBp65 nucleus protein and nuclear translocation on LPS-induced endothelial cell. In conclusion, 2-ME has the effect on monocytes-endothelial cell adhesion, and the mechanism is that inhibiting the role of the nuclear transcription factor NF-_κBp65 and down regulation the expression of VCAM-1. This study will be helpful for further clarify the mechanism of 2-ME anti-AS, and can be used as a new type of clinical drug development.

Effect of a new PPAR-gamma agonist, lobeglitazone, on neointimal formation after balloon injury in rats and the development of atherosclerosis

ObjectiveThe ligand-activated transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) is a key factor in adipogenesis, insulin sensitivity, and cell cycle regulation. Activated PPARγ might also have anti-inflammatory and antiatherogenic properties. We tested whether lobeglitazone, a new PPARγ agonist, might protect against atherosclerosis. MethodsA rat model of balloon injury to the carotid artery, and high-fat, high-cholesterol diet-fed apolipoprotein E gene knockout (ApoE−/−) mice were studied. ResultsAfter the balloon injury, lobeglitazone treatment (0.3 and 0.9 mg/kg) caused a significant decrease in the intima-media ratio compared with control rats (2.2 ± 0.9, 1.8 ± 0.8, vs. 3.3 ± 1.2, P < 0.01). Consistent with this, in ApoE−/− mice fed a high-fat diet, lobeglitazone treatment (1, 3, and 10 mg/kg) for 8 weeks reduced atherosclerotic lesion sizes in the aorta compared with the control mice in a dose-dependent manner. Treatment of vascular smooth muscle cells with lobeglitazone inhibited proliferation and migration and blocked the cell cycle G0/G1 to S phase progression dose-dependently. In response to lobeglitazone, tumor necrosis factor alpha (TNFα)-induced monocyte-endothelial cell adhesion was decreased by downregulating the levels of adhesion molecules. TNFα-induced nuclear factor kappa-B (NF-κB) p65 translocation into the nucleus was also blocked in endothelial cells. Insulin resistance was decreased by lobeglitazone treatment. Circulating levels of high sensitivity C-reactive protein and monocyte chemoattractant protein-1 were decreased while adiponectin levels were increased by lobeglitazone in the high-fat diet-fed ApoE−/− mice. ConclusionLobeglitazone has antiatherosclerotic properties and has potential for treating patients with diabetes and cardiovascular risk.

CD100 and plexins B2 and B1 mediate monocyte-endothelial cell adhesion and might take part in atherogenesis.

Leukocyte migration is essential for the function of the immune system. Their recruitment from the vessels to the tissues involves sequential molecular interactions between leukocytes and endothelial cells (ECs). Many adhesion molecules involved in this process have already been described. However, additional molecules may be important in this interaction, and here we explore the potential role for CD100 and plexins in monocyte-EC binding. CD100 was shown to be involved in platelet-endothelial cell interaction, an important step in atherogenesis and thrombus formation. In a recent work we have described CD100 expression in monocytes and in macrophages and foam cells of human atherosclerotic plaques. In the present work, we have identified plexin B2 as a putative CD100 receptor in these cells. We have detected CD100 expression in the endothelium as well as in in vitro cultured endothelial cells. Blocking of CD100, plexin B1 and/or B2 in adhesion experiments have shown that both CD100 and plexins act as adhesion molecules involved in monocyte-endothelial cell binding. This effect may be mediated by CD100 expressed in both cell types, probably coupled to the receptors endothelial plexin B1 and monocytic plexin B2. These results can bring new insights about a possible biological activity of CD100 in monocyte adhesion and atherosclerosis, as well as a future candidate for targeting therapeutics.

Systems Biology Reveals Cigarette Smoke-Induced Concentration-Dependent Direct and Indirect Mechanisms That Promote Monocyte-Endothelial Cell Adhesion.

Cigarette smoke (CS) affects the adhesion of monocytes to endothelial cells, a critical step in atherogenesis. Using an in vitro adhesion assay together with innovative computational systems biology approaches to analyze omics data, our study aimed at investigating CS-induced mechanisms by which monocyte-endothelial cell adhesion is promoted. Primary human coronary artery endothelial cells (HCAECs) were treated for 4 h with (1) conditioned media of human monocytic Mono Mac-6 (MM6) cells preincubated with low or high concentrations of aqueous CS extract (sbPBS) from reference cigarette 3R4F for 2 h (indirect treatment, I), (2) unconditioned media similarly prepared without MM6 cells (direct treatment, D), or (3) freshly generated sbPBS (fresh direct treatment, FD). sbPBS promoted MM6 cells-HCAECs adhesion following I and FD, but not D. In I, the effect was mediated at a low concentration through activation of vascular inflammation processes promoted in HCAECs by a paracrine effect of the soluble mediators secreted by sbPBS-treated MM6 cells. Tumor necrosis factor α (TNFα), a major inducer, was actually shed by unstable CS compound-activated TNFα-converting enzyme. In FD, the effect was triggered at a high concentration that also induced some toxicity. This effect was mediated through an yet unknown mechanism associated with a stress damage response promoted in HCAECs by unstable CS compounds present in freshly generated sbPBS, which had decayed in D unconditioned media. Aqueous CS extract directly and indirectly promotes monocytic cell-endothelial cell adhesion in vitro via distinct concentration-dependent mechanisms.

Hydrogen-Rich Medium Attenuated Lipopolysaccharide-Induced Monocyte-Endothelial Cell Adhesion and Vascular Endothelial Permeability via Rho-Associated Coiled-Coil Protein Kinase.

Sepsis is the leading cause of death in critically ill patients. In recent years, molecular hydrogen, as an effective free radical scavenger, has been shown a selective antioxidant and anti-inflammatory effect, and it is beneficial in the treatment of sepsis. Rho-associated coiled-coil protein kinase (ROCK) participates in junction between normal cells, and regulates vascular endothelial permeability. In this study, we used lipopolysaccharide to stimulate vascular endothelial cells and explored the effects of hydrogen-rich medium on the regulation of adhesion of monocytes to endothelial cells and vascular endothelial permeability. We found that hydrogen-rich medium could inhibit adhesion of monocytes to endothelial cells and decrease levels of adhesion molecules, whereas the levels of transepithelial/endothelial electrical resistance values and the expression of vascular endothelial cadherin were increased after hydrogen-rich medium treatment. Moreover, hydrogen-rich medium could lessen the expression of ROCK, as a similar effect of its inhibitor Y-27632. In addition, hydrogen-rich medium could also inhibit adhesion of polymorphonuclear neutrophils to endothelial cells. In conclusion, hydrogen-rich medium could regulate adhesion of monocytes/polymorphonuclear neutrophils to endothelial cells and vascular endothelial permeability, and this effect might be related to the decreased expression of ROCK protein.