THP-1 Monocyte Adhesion Research Articles

Eikenella corrodens lipopolysaccharide stimulates the pro-atherosclerotic response in human coronary artery endothelial cells and monocyte adhesion.

Eikenella corrodens is a gram-negative bacterium, and although primarily associated with periodontal infections or infective endocarditis, it has been identified in coronary atheromatous plaques. The effect of its lipopolysaccharide (LPS) on human coronary artery endothelial cells (HCAECs) is unknown. Our aim was to examine the mechanism underlying the inflammatory response in HCAECs stimulated with E.corrodens-LPS and to evaluate monocyte adhesion. Endothelial responses were determined by measuring the levels of chemokines and cytokines using flow cytometry. The surface expression of intercellular adhesion molecule 1 (ICAM-1) was determined using a cell-based ELISA, and the adhesion of THP-1 monocytes to HCAECs was also monitored. The involvement of toll-like receptors (TLRs) 2 and 4 was examined using TLR-neutralizing antibodies, and activation of extracellular signal-regulated kinase (ERK)1/2 and nuclear factor-kappa B (NF-κB) p65 were measured by western blotting and ELISA, respectively. Eikenellacorrodens-LPS increased secretion of interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (GM-CSF), and expression of ICAM-1 on the surface of HCAECs, consistent with the increased adhesion of THP-1 cells. Moreover, E.corrodens-LPS interacted with TLR4, a key receptor able to maintain the levels of IL-8, MCP-1, and GM-CSF in HCAECs. Phosphorylation of ERK1/2 and activation of NF-κB p65 were also increased. The results indicate that E.corrodens-LPS activates HCAECs through TLR4, ERK, and NF-κB p65, triggering a pro-atherosclerotic endothelial response and enhancing monocyte adhesion.

Intermittent Hypoxia Enhances THP-1 Monocyte Adhesion and Chemotaxis and Promotes M1 Macrophage Polarization via RAGE.

Intermittent hypoxia (IH) that resulted from obstructive sleep apnea (OSA) has been found to be a risk factor of coronary artery disease. IH and the receptor for advanced glycation end products (RAGE) expression are known to activate monocyte/macrophage and associated with atherosclerosis development, while their effects on monocyte adhesion, chemotaxis to the endothelium, and macrophage polarization remain unknown. In the present study, RAGE in THP-1 monocytes was inhibited by shRNA lentiviral particles, followed by exposure to IH. Cell adhesion assay, transwell migration assay, and macrophage polarization assays were performed to study the effects of IH and RAGE. The mRNA and protein expression levels were investigated by RT/real-time PCR and western blot analysis, respectively. We found that IH increased RAGE expression and activated NF-кB signalling in THP-1 monocytes. The results also revealed that IH enhanced the MCP-1-mediated THP-1 monocyte adhesion and chemotaxis and promoted macrophage polarization toward a proinflammatory phenotype, which was mediated by RAGE activity. Additionally, inhibition of chemokine receptor type 2 (CCR2) suppressed the IH-induced monocyte adhesion and chemotaxis. These results demonstrated a potential role of monocyte adhesion, chemotaxis, and macrophage polarization in the development cardiovascular diseases induced by IH and identified that RAGE could be a promising therapeutic target to prevent atherosclerosis in patients with OSA.

Association of serum adropin with the presence of atrial fibrillation and atrial remodeling.

Adropin, a newly identified regulatory protein encoded by Enho gene, suppressed tumor necrosis factor α-induced THP1 monocyte adhesion to human umbilical vein endothelial cells. In addition, inflammation is demonstrated to be involved in the mechanism of atrial fibrillation (AF). Atrial remodeling is correlated with the persistence and progression of AF. Adropin is hypothesized to correlated with AF and atrial remodeling. This study aims to determine the correlation of serum adropin and the presence of AF and remodeling. This study consisted of 344 AF patients and 210 healthy controls. AF patients were then divided into three subgroups of paroxysmal AF, persistent AF, and permanent AF. Serum adropin concentrations were examined using enzyme-linked immunosorbent assay method. Left atrial diameter (LAD) was measured to evaluate atrial remodeling. Decreased serum adropin concentrations were found in AF patients compared with healthy controls. Logistic regression analysis confirmed that serum adropin was inversely associated with the presence of AF (OR 0.218, 95% CI 0.15-0.316; P<0.001). Permanent AF patients had significantly reduced serum adropin concentrations compared with persistent and paroxysmal AF patients. There were decreased serum adropin concentrations in persistent AF group than those in paroxysmal AF group. Simple linear regression analyses showed that serum adropin in AF patients were negatively correlated with BMI, SBP, and LAD. Multiple stepwise regression analysis showed that LAD remained to be inversely associated with serum adropin (β=0.2, P=0.010). Serum adropin concentrations are inversely correlated with the presence of AF and atrial remodeling.

Different involvement of the MAPK family in inflammatory regulation in human pulmonary microvascular endothelial cells stimulated with LPS and IFN-γ

Pulmonary endothelial injury is central in the pathogenesis of acute lung injury (ALI). The MAPK signaling cascades are generally thought to be involved in the molecular mechanism underlying the ALI development, but their roles in pulmonary endothelial injury is poorly understood. We thus examined the involvement of the MAPK family member in inflammatory responses of human pulmonary microvascular endothelial cells (HPMVECs) stimulated with LPS and IFN-γ. HPMVECs were found to exhibit the upregulation of expression of Toll-like receptor 4 by IFN-γ, resulting in potentiation of inflammatory cytokine release by LPS stimulation. All MAPKs, ERK1/2, JNK, and p38, were activated by simultaneous stimulation with LPS/IFN-γ. JNK activation in cells stimulated with LPS/IFN-γ was significantly potentiated by the two different p38 inhibitors, SB203580 and RWJ67657, suggesting the negative regulation of JNK activation by p38 in HPMVECs. The mRNA and protein expression levels of ICAM-1 were eliminated by the JNK inhibitor, suggesting that ICAM-1 expression is positively regulated by JNK. The p38 inhibitor significantly enhanced ICAM-1 expression. ERK1/2 activation was not responsible for the LPS/IFN-γ-induced ICAM-1 upregulation in HPMVECs. THP-1 monocyte adhesion to HPMVECs under LPS/IFN-γ stimulation was inhibited by the JNK inhibitor and enhanced by the p38 inhibitor. We conclude that, in HPMVECs stimulated with LPS/IFN-γ, JNK mediates ICAM-1 expression that can facilitate leukocyte adherence and transmigration, while p38 MAPK negatively regulates the upregulation of ICAM-1 through inhibition of JNK activation.

NaoXinTong Capsule Inhibits Carrageenan-Induced Thrombosis in Mice.

Formation of thrombosis is mainly associated with dysfunctions of endothelial cells. NaoXinTong capsule (NXT), a traditional Chinese medicine, has been demonstrated multiple protective effects on vascular systems. However, it is unknown the effect of NXT on thrombosis. In this study, we determined whether NXT can inhibit carrageenan-induced thrombosis and the underlying mechanisms. Two days after carrageenan injection, severe thrombi were found in blood vessels of mouse tail and liver. By contrast, thrombi were substantially reduced by NXT treatment, and the reduction was associated with reduced serum tumor necrosis factor α and P-selectin levels. In vitro, NXT reduced lipopolysaccharide-activated adhesion of THP-1 monocytes to human umbilical vein endothelial cells (HUVECs) by inhibiting expression of adhesion molecules and interleukin 6, and reducing production of mitochondrial superoxide that is related to activation of antioxidant enzymes expression. NXT also reduced oxidized low-density lipoprotein-activated adhesion of platelets to HUVECs. In addition, NXT protected HUVECs against clopidogrel-induced cell death by inhibiting expression of tumor necrosis factor-like cytokine 1A and activating expression of vascular endothelial growth factor α. Taken together, our study indicates the potential application of NXT in antithrombosis by multiple antithrombotic functions.

Fisetin inhibits the generation of inflammatory mediators in interleukin-1β–induced human lung epithelial cells by suppressing the NF-κB and ERK1/2 pathways

Fisetin, a flavone that can be isolated from fruits and vegetables, has anti-tumor and anti-oxidative properties and ameliorates airway hyperresponsiveness in asthmatic mice. This study investigated whether fisetin can suppress the expression of inflammatory mediators and intercellular adhesion molecule 1 (ICAM-1) in A549 human lung epithelial cells that were stimulated with interleukin-1β (IL-1β) to induce inflammatory responses. A549 cells were treated with fisetin (3–30 μM) and then with IL-1β. Fisetin significantly inhibited COX-2 expression and reduced prostaglandin E2 production, and it suppressed the levels of IL-8, CCL5, monocyte chemotactic protein 1, tumor necrosis factor α, and IL-6. Fisetin also significantly attenuated the expression of chemokine and inflammatory cytokine genes and decreased the expression of ICAM-1, which mediates THP-1 monocyte adhesion to inflammatory A549 cells. Fisetin decreased the translocation of nuclear transcription factor kappa-B (NF-κB) subunit p65 into the nucleus and inhibited the phosphorylation of proteins in the ERK1/2 pathway. Co-treatment of IL-1β–stimulated A549 cells with ERK1/2 inhibitors plus fisetin reduced ICAM-1 expression. Furthermore, fisetin significantly increased the effects of the protective antioxidant pathway by promoting the expression of nuclear factor erythroid-2-related factor-2 and heme oxygenase 1. Taken together, these data suggest that fisetin has anti-inflammatory effects and that it suppresses the expression of chemokines, inflammatory cytokines, and ICAM-1 by suppressing the NF-κB and ERK1/2 signaling pathways in IL-1β–stimulated human lung epithelial A549 cells.

Abstract 257: Apabetalone (RVX-208) Reduces Pathologic Cell-Cell Adhesion and Expression of Key Vascular Inflammation Markers in Monocytes, Endothelial Cells and Mouse Aorta

Apabetalone (RVX-208) is a bromodomain &amp; extraterminal (BET) protein inhibitor, an epigenetic modifier of gene expression, currently in a phase 3 major adverse cardiac events outcomes trial in post-acute coronary syndrome patients with type 2 diabetes mellitus (DM) (BETonMACE). CVD patients enrolled in phase 2b trials (ASSERT, SUSTAIN and ASSURE) demonstrated a 44% relative risk reduction in cardiovascular disease (CVD) events (Nicholls et al. 2017). In CVD and DM, elevated circulating cytokines potentiate vascular inflammation (VI) through recruitment of leukocytes to the vascular endothelium, which contributes to atherosclerosis and plaque rupture. Previous studies demonstrated that apabetalone has potent anti-inflammatory effects on human aortic endothelial cells (HAEC) and macrophage-like U937 cells. Here we show that TNFα stimulation induced significant adhesion of THP-1 monocytes to inflamed endothelial cells, an outcome reversed by apabetalone treatment under both static (human umbilical vein endothelial cells - HUVEC) and flow (HAEC) conditions. Mechanistically, apabetalone suppressed the TNFα and IL-1β-induced expression of mRNAs of multiple endothelial cell adhesion molecules (CD44, E-selectin, VCAM-1 and MCP-1) and inflammatory cytokines (IL-6, IL-8, IL-1β, and CSF2). Monocytes also respond to TNFα stimulation with an upregulation of inflammatory and adhesion marker expression. In THP-1 cells, apabetalone treatment significantly reduced mRNA expression of CCR1, CCR2, IL-1β, MCP-1, MYD88, TLR4, TNFα, and VLA-4. In the diet-induced obesity (DIO) mouse model that mimics metabolic syndrome, treatment with apabetalone, administered at 150 mg/kg BID for the last 16 weeks of a 22 week study, downregulated aortic adhesion markers (E-selectin and ICAM) and markers of infiltrating immune cells (CCR2 and CD11b). In summary, treatment with apabetalone causes transcriptional changes in monocytes and endothelial cells that translate into a reduction in adhesion under inflammatory conditions. We hypothesize that downregulation of VI by apabetalone may contribute to the reduction in CVD events observed in phase 2 studies. This hypothesis is currently being tested in the ongoing BETonMACE phase 3 trial.

Abstract 623: Activating Liver X Receptors Alters Endothelial Progenitor Cell Differentiation and Enhances the Release of Factors that Reduce Atherosclerosis

The liver x receptors (LXRα/β) are nuclear receptors that are highly expressed in macrophages and endothelial progenitor cells (EPCs). EPCs are bone marrow (BM) derived cells that facilitate endothelial repair through the secretion of factors. The importance of LXRs in preventing atherosclerosis has been attributed to promoting cholesterol efflux from macrophages. However, a role for EPCs in the LXR response has not been explored. We hypothesized that LXR activation in EPCs contributes to their anti-atherogenic effects by preventing endothelial activation during atherogenesis. In the Lxr -/- mice, circulating EPCs were decreased by 27% compared to WT, while myeloid populations were increased. Isolated hematopoietic stem cells treated with LXR agonist (1μM GW3965) ex vivo showed a 36% increase in the number of EPC colonies formed, while myeloid colonies were decreased by 25%. GW3965 treatment during EPC differentiation resulted in a 76% decrease in endothelial lineage marker ( Cd144 and Vegfr2 ) expression. To investigate endothelial cell activation, activated human umbilical vein endothelial cells were incubated with the conditioned media (CM) from GW3965-treated WT EPCs, which reduced THP-1 monocyte adhesion by 36% compared to the CM from vehicle treatment. To determine the effects of the EPC-derived CM in vivo , Ldlr -/- mice were injected with concentrated CM bi-weekly over an 8-week period, which decreased aortic sinus lesion area by 52% compared to the media control injections. Taken together, the loss of LXRs perturbed hematopoietic balance in vivo , while the activation of LXRs were associated with enhanced ex vivo EPC formation. Furthermore, LXR activation altered EPC differentiation and secretome resulting in decreased in vitro endothelial-monocyte adhesion. When administered to atherosclerotic-prone mice, these secreted factors reduced lesion formation in the aortic sinus. These data suggest that LXRs reduce the development of atherosclerosis in part through the release of EPC-derived factors that can reduce endothelial activation.

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.

Vascular Semaphorin 7A Upregulation by Disturbed Flow Promotes Atherosclerosis Through Endothelial β1 Integrin.

Accumulating evidence suggests a role of semaphorins in vascular homeostasis. Here, we investigate the role of Sema7A (semaphorin 7A) in atherosclerosis and its underlying mechanism. Using genetically engineered Sema7A-/-ApoE-/- mice, we showed that deletion of Sema7A attenuates atherosclerotic plaque formation primarily in the aorta of ApoE-/- mice on a high-fat diet. A higher level of Sema7A in the atheroprone lesser curvature suggests a correlation of Sema7A with disturbed flow. This notion is supported by elevated Sema7A expression in human umbilical venous endothelial cells either subjected to oscillatory shear stress or treated with the PKA (protein kinase A)/CREB (cAMP response element-binding protein) inhibitor H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide·2HCl hydrate). Further studies using the partial carotid artery ligation model showed that disturbed flow in the left carotid artery of Sema7A+/+ApoE-/- mice promoted the expression of endothelial Sema7A and cell adhesion molecules, leukocyte adhesion, and plaque formation, whereas such changes were attenuated in Sema7A-/-ApoE-/- mice. Further studies showed that blockage of β1 integrin, a known Sema7A receptor, or inhibition of FAK (focal adhesion kinase), MEK1/2 (mitogen-activated protein kinase kinase 1/2), or NF-κB (nuclear factor-κB) significantly reduced the expression of cell adhesion molecules and THP-1 (human acute monocytic leukemia cell line) monocyte adhesion in Sema7A-overexpressing human umbilical venous endothelial cells. Studies using chimeric mice suggest that vascular, most likely endothelial, Sema7A plays a major role in atherogenesis. Our findings indicate a significant role of Sema7A in atherosclerosis by mediating endothelial dysfunction in a β1 integrin-dependent manner.

The adverse vascular effects of multi-walled carbon nanotubes (MWCNTs) to human vein endothelial cells (HUVECs) in vitro: role of length of MWCNTs

BackgroundIncreasing evidences indicate that exposure to multi-walled carbon nanotubes (MWCNTs) could induce adverse vascular effects, but the role of length of MWCNTs in determining the toxic effects is less studied. This study investigated the adverse effects of two well-characterized MWCNTs to human umbilical vein endothelial cells (HUVECs).MethodsThe internalization and localization of MWCNTs in HUVECs were examined by using transmission electron microscopy (TEM). The cytotoxicity of MWCNTs to HUVECs was assessed by water soluble tetrazolium-8 (WST-8), lactate dehydrogenase (LDH) and neutral red uptake assays. Oxidative stress was indicated by the measurement of intracellular glutathione (GSH) and reactive oxygen species (ROS). ELISA was used to determine the release of inflammatory cytokines. THP-1 monocyte adhesion to HUVECs was also measured. To indicate the activation of endoplasmic reticulum (ER) stress, the expression of ddit3 and xbp-1s was measured by RT-PCR, and BiP protein level was measured by Western blot.ResultsTransmission electron microscopy observation indicates the internalization of MWCNTs into HUVECs, with a localization in nuclei and mitochondria. The longer MWCNTs induced a higher level of cytotoxicity to HUVECs compared with the shorter ones. Neither of MWCNTs significantly promoted intracellular ROS, but the longer MWCNTs caused a higher depletion of GSH. Exposure to both types of MWCNTs significantly promoted THP-1 adhesion to HUVECs, accompanying with a significant increase of release of interleukin-6 (IL-6) but not tumor necrosis factor α (TNFα), soluble ICAM-1 (sICAM-1) or soluble VCAM-1 (sVCAM-1). Moreover, THP-1 adhesion and release of IL-6 and sVCAM-1 induced by the longer MWCNTs were significantly higher compared with the responses induced by the shorter ones. The biomarker of ER stress, ddit3 expression, but not xbp-1s expression or BiP protein level, was significantly induced by the exposure of longer MWCNTs.ConclusionsCombined, these results indicated length dependent toxic effects of MWCNTs to HUVECs in vitro, which might be associated with oxidative stress and activation of ER stress.

Lunasin abrogates monocytes to endothelial cells

The adherence of monocytes to endothelial cells plays a causal role in the early development of atherosclerosis and is driven by several inflammatory stimuli, which includes oxidized low-density lipoprotein (ox-LDL). Lunasin, a natural peptide identified in soybean seeds, soy-derived food products, other grains and herbal plants, has been found to exert numerous biological activities, including anti-inflammatory and antioxidant properties. However, little is known regarding the mechanism of action of lunasin in ox-LDL-induced endothelial inflammation. The results of the present study indicate that lunasin significantly ameliorated ox-LDL-induced adhesion of THP-1 monocytes to the surface of human umbilical vein endothelial cells (HUVECs). Lunasin also suppressed expression of the adhesion molecules VCAM-1 and E-selectin, but not ICAM-1. Notably, the inhibitory mechanism of lunasin is associated with its stimulatory effects on expression of the KLF2 transcriptional factor. In addition, lunasin treatment could reverse the effects of ox-LDL on the expression of eNOS and PAI-1, the direct target genes of KLF2. Mechanistically, it was proven that the MEK5/ERK5 pathway mediates the effects of lunasin on KLF2 expression. Taken together, the results of this study suggest that dietary or supplementary intake of lunasin may have a prophylactic or therapeutic capacity in cardiovascular diseases such as atherosclerosis.

MicroRNA-214-3p: A link between autophagy and endothelial cell dysfunction in atherosclerosis.

Endothelial cell injury assumes a fundamental part in the pathogenesis of atherosclerosis, and endothelial cell autophagy has protective effects on the development of atherosclerosis, although the underlying molecular regulation mechanism is indistinct. This study aimed to investigate whether microRNA-214-3p (miR-214-3p) is involved in the endothelial cell autophagy regulation of atherosclerosis. We utilized ApoE-/- mice provided with a high-fat diet (HFD) as atherosclerosis model. We analysed the level of miR-214-3p and the levels of autophagy-related protein 5 (ATG5) and autophagy-related protein 12 (ATG12) in the purified CD31+ endothelial cells from mouse aorta. Bioinformatics analysis and a dual-luciferase reporter assay were performed to confirm the binding target of miR-214-3p. In vitro study, human umbilical vein endothelial cells (HUVECs) were transfected with miR-214-3p mimics/inhibitor and stimulated with 100 μg/mL oxidized low-density lipoprotein (ox-LDL) for 12 hours to initiate a stress-repairing autophagic process. In mouse models, we identified an inverse correlation between miR-214-3p, ATG5 and ATG12. We observed that in young HUVECs, ox-LDL-initiated autophagy was repressed by miR-214-3p overexpression, as evaluated by autophagic protein analysis, microtubule-associated protein 1 light chain 3B-II (LC3B-II) immunofluorescence assay and transmission electron microscopy (TEM). Also, miR-214-3p promoted ox-LDL accumulation in HUVECs and THP-1 monocyte adhesion. Conversely, in old HUVECs, suppression of miR-214-3p preserved the ability to initiate a protective autophagy reaction to the ox-LDL stimulation. miR-214-3p regulates ox-LDL-initiated autophagy in HUVECs by directly targeting the 3'UTR of ATG5 and may have a suitable role in the pathogenesis of atherosclerosis.

In vitro effect of flaxseed oil and α-linolenic acid against the toxicity of lipopolysaccharide (LPS) to human umbilical vein endothelial cells.

Consumption of α-linolenic acid (ALA)-rich flaxseed oil is a possible way to supplement ω-3 polyunsaturated fatty acids, but the beneficial effects to cardiovascular systems are still controversial. In this study, human umbilical vein endothelial cells (HUVECs) treated with 100ng/mL lipopolysaccharide (LPS) were used as the in vitro model and the potential beneficial effects of well-characterized flaxseed oil and ALA were studied. Exposure of HUVECs to LPS for 24h significantly promoted inflammatory response as release of interleukin 6 (IL-6), soluble intercellular cell adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule 1 (sVCAM-1) as well as adhesion of THP-1 monocytes, but did not induce cytotoxicity or oxidative stress. ALA, but not flaxseed oil, significantly reduced LPS-induced release of sICAM-1 and sVCAM-1, without effect on THP-1 adhesion. No radical scavenging activity was observed after flaxseed oil or ALA exposure. Rather, ALA at high concentrations promoted intracellular superoxide associated with damages to lysosomes, which was not observed in flaxseed oil exposed cells. These results indicated that ALA at high concentrations could inhibit inflammatory responses in LPS-treated HUVECs in vitro but might also promote a modest effect in cytotoxicity and oxidative stress.

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.

Telmisartan mitigates hyperglycemia-induced vascular inflammation by increasing GSK3β-Ser9 phosphorylation in endothelial cells and mouse aortas

Telmisartan, an angiotensin II type 1 receptor blocker (ARB), attenuates hyperglycemia-aggravated vascular inflammation by decreasing IκB kinase β (IKKβ) expression in endothelial cells. Because glycogen synthase 3β (GSK3β) is involved in inflammatory process by regulating nuclear factor-κB (NF-κB) activity, we investigated whether GSK3β mediates telmisartan-ameliorated vascular inflammation in hyperglycemia-treated endothelial cells and high-fat diet (HFD)-fed mice. Telmisartan remarkably induced GSK3β-Ser9 phosphorylation in hyperglycemia-treated endothelial cells that accompanied a decrease in hyperglycemia-induced NF-κB p65-Ser536 phosphorylation, vascular cell adhesion molecule-1 (VCAM-1) expression, and THP-1 monocyte adhesion. Ectopic expression of GSK3β-S9A, a constitutively active mutant of GSK3β, significantly restored complete telmisartan-inhibited NF-κB p65-Ser536 phosphorylation, VCAM-1 expression, and THP-1 monocyte adhesion. In addition, it reversed telmisartan-repressed IKKβ expression. Among the ARB, including losartan and fimasartan, only telmisartan increased GSK3β-Ser9 phosphorylation, and telmisartan-induced GSK3β-Ser9 phosphorylation remained unchanged by pretreatment with GW9662, a specific and irreversible peroxisome proliferator-activated receptor γ (PPARγ) antagonist. Finally, in the aortas of HFD-fed mice, telmisartan treatment significantly attenuated HFD-induced upregulation of NF-κB p65-Ser536 phosphorylation, VCAM-1 expression, and IKKβ expression and downregulation of GSK3β-Ser9 phosphorylation. Taken together, our findings demonstrated that telmisartan ameliorates hyperglycemia-exacerbated vascular inflammation, at least in part, by inducing GSK3β-Ser9 phosphorylation, which consequently inhibits IKKβ expression, NF-κB p65-Ser536 phosphorylation, and VCAM-1 expression in a PPARγ-independent manner.

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

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

Ziziphus nummularia Inhibits Inflammation-Induced Atherogenic Phenotype of Human Aortic Smooth Muscle Cells.

Cardiovascular disease (CVD) continues to be the leading cause of death worldwide. Atherosclerosis is a CVD characterized by plaque formation resulting from inflammation-induced insults to endothelial cells, monocytes, and vascular smooth muscle cells (VSMCs). Despite significant advances, current treatments for atherosclerosis remain insufficient, prompting the search for alternative modalities, including herbal medicine. Ziziphus nummularia is an herb commonly used in the amelioration of symptoms associated with many health conditions such as cold, diarrhea, cancer, and diabetes. However, its effect on the inflammation-induced behavior of VSMCs remains unknown. In this study, we sought to determine the effect of the ethanolic extract of Z. nummularia (ZNE) on TNF-α-induced phenotypic changes of human aortic smooth muscle cells (HASMCs). The treatment of HASMCs with ZNE decreased cell proliferation, adhesion to fibronectin, migration, and invasion. ZNE treatment also caused a concentration- and time-dependent reduction in the TNF-α-induced expression of matrix metalloproteases MMP-2 and MMP-9, NF-κB, and cell adhesion molecules ICAM-1 and VCAM-1. Furthermore, ZNE decreased the adhesion of THP-1 monocytes to HASMCs and endothelial cells in a concentration-dependent manner. These data provide evidence for the anti-inflammatory effect of Ziziphus nummularia, along with potential implications for its use as an agent that could ameliorate inflammation-induced atherogenic phenotype of VSMCs in atherosclerosis.

P-Selectin and ICAM-1 synergy in mediating THP-1 monocyte adhesion in hemodynamic flow is length dependent.

The tightly orchestrated recruitment of monocytes, whose progeny are critical to the progression and resolution of various physiological and pathophysiological processes, is implicated in the time course, severity, and resolution of pathology. Using a microfluidic-based cell adhesion assay integrating spatiotemporal analyses and micropatterning of adhesive proteins, we interrogated the effects of adhesive molecule presentation length, which varies in vivo with disease and stage, on THP-1 monocyte cell rolling versus firm adhesion mediated by P-selectin and/or ICAM-1 in hemodynamic flow. Our results indicate that co-presentation of P-selectin and ICAM-1 substantially decreases the length of adhesive substrate required to sustain adhesion in flow and that P-selectin functions synergistically with ICAM-1 to substantially enhance THP-1 firm adhesion. This synergy was found to furthermore correlate with diminished cell rolling velocities and length-enhanced secondary cell capture. Our results suggest pathophysiological ramifications for local remodeling of the inflamed microvascular microenvironment in directing the efficiency of monocyte trafficking.

Evaluation of in vitro toxicity of polymeric micelles to human endothelial cells under different conditions

Polymeric micelles have been extensively studied in the area of antitumor therapy, and more recently explored as nanocarriers for atherosclerosis. These applications of polymeric micelles in biomedicine will increase their contact with human blood vessels. However, few studies have considered the interactions between polymeric micelles and endothelial cells, especially in a complex system. This study used human umbilical vein endothelial cells (HUVECs) as an in vitro model for endothelial cells to investigate the toxic effects of methoxy-poly(ethylene glycol)-poly(d,l-lactide) (MPEG-PLA) based micelles. In addition, an endoplasmic reticulum stress inducer thapsigargin (TG), and a pro-atherogenic stimulus palmitate (PA), were used to co-expose HUVECs to further mimic the responses of diseased endothelial cells to micelle exposure. Overall, up to 200 μg/mL micelles did not significantly induce cytotoxicity, reactive oxygen species (ROS), release of inflammatory mediators in terms of interleukin 6 (IL-6), IL-8 and soluble vascular cell adhesion molecule 1 (sVCAM-1), or adhesion of THP-1 monocytes to HUVECs. TG and PA significantly induced cytotoxicity and THP-1 adhesion as well as modestly promoted the release of IL-6, but did not affect ROS or release of sVCAM-1 and IL-8. Co-exposure of micelles did not significantly enhance the effects of TG and PA to HUVECs, and ANOVA analysis indicated no interaction between concentrations of micelles and the presence of TG/PA. Taken together, these data indicated that micelles are not toxic to HUVECs under different conditions in vitro.