E-selectin mRNA Expression Research Articles

MTOR Inhibitor Rapalink-1 Prevents Ethanol-Induced Senescence in Endothelial Cells.

The cardiovascular risk factors, including smoking, ethanol, and oxidative stress, can induce cellular senescence. The senescent cells increase the expression and release of pro-inflammatory molecules and matrix metalloproteinase (MMPs). These pro-inflammatory molecules and MMPs promote the infiltration and accumulation of inflammatory cells in the vascular tissue, exacerbating vascular tissue inflammation. MMPs damage vascular tissue by degenerating the extracellular matrix. Consequently, these cellular and molecular events promote the initiation and progression of cardiovascular diseases. We used Rapalink-1, an mTOR inhibitor, to block ethanol-induced senescence. Rapalink-1 inhibited oxidative-stress-induced DNA damage and senescence in endothelial cells exposed to ethanol. It attenuated the relative protein expression of senescence marker P21 and improved the relative protein expression of DNA repair protein KU70 and aging marker Lamin B1. It inhibited the activation of NF-κB, MAPKs (P38 and ERK), and mTOR pathway proteins (mTOR, 4EBP-1, and S6). Moreover, Rapalink-1 suppressed ethanol-induced mRNA expression of ICAM-1, E-selectin, MCP-1, IL-8, MMP-2, and TIMP-2. Rapalink-1 also reduced the relative protein expression of MMP-2. In summary, Rapalink-1 prevented senescence, inhibited pro-inflammatory pathway activation, and ameliorated pro-inflammatory molecule expression and MMP-2.

Dienogest does not augment the gene expression of adhesion molecules, MCP-1, and monocyte adherence in human endothelial cells

Objective Medroxyprogesterone acetate (MPA) may increase the risk of atherosclerosis during hormone replacement therapy (HRT); therefore, the effect of progestogens other than MPA on atherosclerotic lesions requires evaluation. Adhesion of monocytes to vascular endothelial cells is an important early step in atherosclerosis progression. MCP-1 is a key chemokine that promotes monocyte migration and adhesion to vascular endothelial cells. In this study, we investigated the effects of dienogest (DNG), an alternative progestogen, on monocyte adhesion and cytokine expression in human umbilical vein endothelial cells (HUVECs). Study Design HUVECs were treated with DNG, natural progesterone, or MPA, followed by interleukin (IL)-1β stimulation. The mRNA expression of adhesion molecules (E-selectin and ICAM-1) and cytokines (MCP-1 and IL-6) was examined using real-time PCR. A flow chamber system was used to examine the effect of DNG on the adhesion of U937 monocytic cells to monolayer HUVECs. Results Unlike MPA, DNG did not alter the mRNA expression of E-selectin, ICAM-1, MCP-1, and IL-6 in HUVECs. Moreover, it did not increase the number of monocytes adhering to HUVECs in the flow chamber system. However, MPA treatment significantly enhanced monocyte adhesion to HUVECs (p < 0.05). Conclusions DNG had no effect on the mRNA expression of adhesion molecules and cytokines in HUVECs, as well as the monocyte adhesion to HUVECs, suggesting that DNG can be explored as an alternative to MPA for HRT.

Lactobacillus plantarum HFY05 Attenuates Carrageenan-Induced Thrombosis in Mice by Regulating NF-κB Pathway-Associated Inflammatory Responses.

In this study, a carrageenan-induced thrombus model was established in mice to observe the ability of Lactobacillus plantarum KFY05 (LP-KFY05) to inhibit thrombosis through an NF-κB-associated pathway. Biochemical analysis, microscopical observations, quantitative polymerase chain reactions (qPCR) and western blot analysis were used to examine relevant serum and tissue indexes, and the composition of intestinal microorganisms was determined by examining the abundance of microorganisms in feces. The results showed that LP-KFY05 could markedly reduce the degree of black tail in thrombotic mice; increase the activated partial thromboplastin time (APTT); and decrease the thrombin time (TT), fibrinogen (FIB) level, and prothrombin time (PT). LP-KFY05 could also reduce tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) levels in sera and renal tissues of thrombotic mice. Hematoxylin and eosin staining showed that LP-KFY05 could alleviate renal tissue lesions and tail vein thrombosis. qPCR results showed that LP-KFY05 could down-regulate nuclear factor kappa-B (NF-κB) p65, IL-6, TNF-α, and interferon γ (IFN-γ) mRNA expression in renal tissues, as well as NF-κB p65, intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin mRNA expression in tail vein vascular tissues of thrombotic mice. Western blot analysis showed that LP-KFY05 also down-regulated NF-κB protein expression in renal and tail vein vascular tissues of thrombotic mice. Lastly, LP-KFY05 increased the abundances of Bacteroidetes, Lactobacillus, and Bifidobacterium, as well as decreased the abundance of Firmicutes. These results show that LP-KFY05 can reduce inflammation and inhibit thrombosis in thrombotic mice, and the effects of high concentrations of LP-KFY05 were most pronounced, which were similar to the effects of dipyridamole.

Apocynum Leaf Extract Suppresses the Progress of Atherosclerosis in Rats via the FKN/SYK/p38 Signal Pathway.

To investigate the antiatherosclerotic effects of flavonoids extracted from Apocynum venetum (AVF) leaves in atherosclerotic rats and the underlying mechanisms, a total of 72 male Wistar rats were randomly divided into six groups: control group, model group, simvastatin group, low-dose AVF group, medium-dose AVF group, and high-dose AVF group. Atherosclerosis in rats was induced with a high-fat diet and an intraperitoneal injection of VD3 once daily for three contiguous days at a total injection dose of 70 U/kg. At the end of the 13th week, total serum cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) contents were measured. The hematoxylin-eosin (HE) staining was applied to evaluate the morphological changes. The ELISA method was used to detect related inflammatory factors and oxidative stress indicators. The corresponding protein expression and the mRNA level were detected by western blot analysis and reverse transcriptase PCR. HE staining showed that the thoracic aorta wall was thickened, and the aortic subendothelial foam cells and lipid vacuoles were reduced in the medium/high-AVF groups. Similarly, the TC, TG, LDL-C, and malondialdehyde (MDA) levels in the model group were significantly higher, but the HDL-C level and superoxide dismutase (SOD) activity were lower than those of the control group, and these effects were ameliorated by treatment with simvastatin or AVF. ELISA results showed that compared with the control group, the model group C-reactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) results were significantly increased, and the medium AVF and high AVF could significantly reduce the expression of related inflammatory factors. The AVF inhibited intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin mRNA and related protein expression in the aorta in atherosclerotic rats. Western blot analysis also showed that AVF can significantly reduce the protein expression of fractalkine (FKN), spleen tyrosine kinase (SYK), and p38 mitogen-activated protein kinase (p38) in the rat aorta. We believe that the AVF can effectively reduce blood lipid levels in rats with atherosclerosis and delay atherosclerotic progression by inhibiting excessive inflammatory factors and inhibiting related adhesion factors. The underlying mechanism may be related to the FKN/SYK/p38 signaling pathway activity. Our results contribute to validating the traditional use of the Apocynum leaf extract in the treatment of atherosclerosis.

α-Conidendrin inhibits the expression of intercellular adhesion molecule-1 induced by tumor necrosis factor-α in human lung adenocarcinoma A549 cells

α-Conidendrin is a lignan isolated from Taxus wallichiana and other species. In the present study, we demonstrated that α-conidendrin inhibited the cell-surface expression of intercellular adhesion molecule-1 (ICAM-1) induced by tumor necrosis factor-α (TNF-α) at an IC50 value of 40–60 μM in human lung adenocarcinoma A549 cells. α-Conidendrin decreased ICAM-1 protein and mRNA expression levels at concentrations of 40–100 μM in TNF-α-stimulated A549 cells. The TNF-α-induced mRNA expression of vascular cell adhesion molecule-1, E-selectin, and cyclooxygenase-2 was also reduced by α-conidendrin. In the TNF-α-induced nuclear factor κB (NF-κB) signaling pathway, α-conidendrin did not influence the translocation of the NF-κB subunit RelA from the cytoplasm to the nucleus at concentrations up to 100 μM. A chromatin immunoprecipitation assay revealed that α-conidendrin at 100 μM reduced the binding of RelA to the ICAM-1 promoter in response to a stimulation with TNF-α. Collectively, these results indicated that α-conidendrin interfered with the DNA binding of RelA to the ICAM-1 promoter, thereby reducing ICAM-1 transcription.

Insights on the anti-inflammatory and antitumor activities of extracts from the marine green alga Codium decorticatum

IntroductionThe Codium genus has been shown to be an important source of marine bioactive compounds for pharmaceutical and nutraceutical applications. The present study was conducted to investigate the anti-inflammatory activity of the n-hexane (Extr 1), dichloromethane (Extr 2) and acetone/methanol (Extr 3) extracts of the green alga Codium decorticatum, as well as their antitumor effects on several cancer cell lines. MethodsIn order to evaluate the anti-inflammatory effect of the extracts of C. decorticatum on the production of the pro-inflammatory proteins, we have undertaken analyses of LPS- and TNF-α-stimulated endothelial cells treated with extracts using enzyme-linked immunosorbent assay (ELISA). Real-time PCR (qPCR) was used to modulate the mRNA expression of inflammatory genes. For the antitumor activity, in vitro cytotoxicity was determined by MTT assay and apoptosis was determined by flow cytometry. ResultsOur results revealed that the Extr 2 and Extr 3 dramatically inhibited the expression of the pro-inflammatory cytokine Interleukin-8 (IL-8) in LPS- and TNF-α-stimulated endothelial cells. We also demonstrated that Extr 2 suppressed the LPS-induced mRNA expression of E-selectin and IL-8. Furthermore, Extr 2 showed cytotoxic activity against the HeLa cell line (IC50 = 37.22 μg/mL), by inducing apoptosis at highest concentrations. ConclusionsThe present data suggest that the seaweed C. decorticatum has the anti-inflammatory properties in endothelial cells, together with the anticancer effect on cervix cancer cells, which may have a potential therapeutic use for inflammatory diseases and cancer. Although, its application as nutraceuticals and functional food could be also considered.

MON-214 1α,25-DIHYDROXYVITAMIN D3 TARGETING OF NF-ΚB SUPPRESSES TNF-Α INDUCED ADHESION MOLECULE EXPRESSION IN HUMAN ENDOTHELIAL CELLS

Vitamin D and its analogues have been documented to be associated with endothelial dysfunction in various diseases. However, the underlying mechanism remains unknown. Here, we conducted an in vitro study to evaluate the effect of 1α,25-dihydroxyvitamin D3, the active form of vitamin D3, on adhesion molecule expression in human endothelial cells. The possible mechanism involved in this process was also explored. Human umbilical vein cells (HUVECs) were cultured and treated according to the experiment requirement. Western Blot and RT-PCR were used to evaluate the expression of vascular cell adhesion molecular-1 (VCAM-1) and E-selectin. ChIP assay, immunofluorescence, Western Blot and co-immunoprecipitation were used to assess the effect of 1α,25-dihydroxyvitamin D3 on NF-κB signaling. 1α,25-dihydroxyvitamin D3 inhibited VCAM-1 and E-selectin mRNA and protein expression after TNF-α stimulation. ChIP assay showed that TNF-α increased the p65 binding to the promoter of VCAM-1 and E-selectin, which was suppressed by 1α,25-dihydroxyvitamin D3. 1α,25-dihydroxyvitamin D3 affected TNF-α induced IκBα phosphorylation and p65 NF-κB activation, leading to an inhibition of p65 nuclear translocation. These effects were reversed by a specific vitamin D receptor siRNA (VDR-siRNA). Co-immunoprecipitation revealed that 1α,25-dihydroxyvitamin D3 induced an increased binding of VDR to p65, which inhibited the ability of p65 binding to target gene promoters. 1,25-dihydroxyvitamin D3 suppresses TNF-α induced adhesion molecule expression in human endothelial cells by blocking the NF-κB pathway, and this was VDR dependent.

Oolong Tea Extract and Citrus Peel Polymethoxyflavones Reduce Transformation of l-Carnitine to Trimethylamine-N-Oxide and Decrease Vascular Inflammation in l-Carnitine Feeding Mice.

Carnitine, a dietary quaternary amine mainly from red meat, is metabolized to trimethylamine (TMA) by gut microbiota and subsequently oxidized to trimethylamine-N-oxide (TMAO) by host hepatic enzymes, flavin monooxygenases (FMOs). The objective of this study aims to investigate the effects of flavonoids from oolong tea and citrus peels on reducing TMAO formation and protecting vascular inflammation in carnitine-feeding mice. The results showed that mice treated with 1.3% carnitine in drinking water significantly (p < 0.05) increased the plasma levels of TMAO compared to control group, whereas the plasma TMAO was remarkedly reduced by flavonoids used. Meanwhile, these dietary phenolic compounds significantly (p < 0.05) decreased hepatic FMO3 mRNA levels compared to carnitine only group. Additionally, oolong tea extract decreased mRNA levels of vascular inflammatory markers such as tissue necrosis factor-alpha (TNF-α), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. Polymethoxyflavones significantly lowered the expression of VCAM-1 and showed a decreasing trend in TNF-α and E-selectin mRNA expression compared to the carnitine group. Genus-level analysis of the gut microbiota in the cecum showed that these dietary phenolic compounds induced an increase in the relative abundances of Bacteroides. Oolong tea extract-treated group up-regulated Lactobacillus genus, compared to the carnitine only group. Administration of polymethoxyflavones increased Akkermansia in mice.

Olive Leaf Extract Attenuates Inflammatory Activation and DNA Damage in Human Arterial Endothelial Cells.

Olive leaf extract (OLE) is used in traditional medicine as a food supplement and as an over-the-counter drug for a variety of its effects, including anti-inflammatory and anti-atherosclerotic ones. Mechanisms through which OLE could modulate these pathways in human vasculature remain largely unknown. Serum amyloid A (SAA) plays a causal role in atherosclerosis and cardiovascular diseases and induces pro-inflammatory and pro-adhesive responses in human coronary artery endothelial cells (HCAEC). Within this study we explored whether OLE can attenuate SAA-driven responses in HCAEC. HCAEC were treated with SAA (1,000 nM) and/or OLE (0.5 and 1 mg/ml). The expression of adhesion molecules VCAM-1 and E-selectin, matrix metalloproteinases (MMP2 and MMP9) and microRNA 146a, let-7e, and let-7g (involved in the regulation of inflammation) was determined by qPCR. The amount of secreted IL-6, IL-8, MIF, and GRO-α in cell culture supernatants was quantified by ELISA. Phosphorylation of NF-κB was assessed by Western blot and DNA damage was measured using the COMET assay. OLE decreased significantly released protein levels of IL-6 and IL-8, as well as mRNA expression of E-selectin in SAA-stimulated HCAEC and reduced MMP2 levels in unstimulated cells. Phosphorylation of NF-κB (p65) was upregulated in the presence of SAA, with OLE significantly attenuating this SAA-induced effect. OLE stabilized SAA-induced upregulation of microRNA-146a and let-7e in HCAEC, suggesting that OLE could fine-tune the SAA-driven activity of NF-κB by changing the microRNA networks in HCAEC. SAA induced DNA damage and worsened the oxidative DNA damage in HCAEC, whereas OLE protected HCAEC from SAA- and H2O2-driven DNA damage. OLE significantly attenuated certain pro-inflammatory and pro-adhesive responses and decreased DNA damage in HCAEC upon stimulation with SAA. The reversal of SAA-driven endothelial activation by OLE might contribute to its anti-inflammatory and anti-atherogenic effects in HCAEC.

LSEC Fenestrae Are Preserved Despite Pro-inflammatory Phenotype of Liver Sinusoidal Endothelial Cells in Mice on High Fat Diet.

Healthy liver sinusoidal endothelial cells (LSECs) maintain liver homeostasis, while LSEC dysfunction was suggested to coincide with defenestration. Here, we have revisited the relationship between LSEC pro-inflammatory response, defenestration, and impairment of LSEC bioenergetics in non-alcoholic fatty liver disease (NAFLD) in mice. We characterized inflammatory response, morphology as well as bioenergetics of LSECs in early and late phases of high fat diet (HFD)-induced NAFLD. LSEC phenotype was evaluated at early (2–8 week) and late (15–20 week) stages of NAFLD progression induced by HFD in male C57Bl/6 mice. NAFLD progression was monitored by insulin resistance, liver steatosis and obesity. LSEC phenotype was determined in isolated, primary LSECs by immunocytochemistry, mRNA gene expression (qRT-PCR), secreted prostanoids (LC/MS/MS) and bioenergetics (Seahorse FX Analyzer). LSEC morphology was examined using SEM and AFM techniques. Early phase of NAFLD, characterized by significant liver steatosis and prominent insulin resistance, was related with LSEC pro-inflammatory phenotype as evidenced by elevated ICAM-1, E-selectin and PECAM-1 expression. Transiently impaired mitochondrial phosphorylation in LSECs was compensated by increased glycolysis. Late stage of NAFLD was featured by prominent activation of pro-inflammatory LSEC phenotype (ICAM-1, E-selectin, PECAM-1 expression, increased COX-2, IL-6, and NOX-2 mRNA expression), activation of pro-inflammatory prostaglandins release (PGE2 and PGF2α) and preserved LSEC bioenergetics. Neither in the early nor in the late phase of NAFLD, were LSEC fenestrae compromised. In the early and late phases of NAFLD, despite metabolic and pro-inflammatory burden linked to HFD, LSEC fenestrae and bioenergetics are functionally preserved. These results suggest prominent adaptive capacity of LSECs that might mitigate NAFLD progression.

Equilin in conjugated equine estrogen increases monocyte-endothelial adhesion via NF-κB signaling.

The adhesion of monocytes to endothelial cells, which is mediated by adhesion molecules, plays a crucial role in the onset of atherosclerosis. Conjugated equine estrogen, which is widely used for estrogen-replacement therapy, contains both estrone sulfate and various nonhuman estrogens, including equilin. To investigate the association between various estrogen types and atherosclerosis risk, we examined their effect on adhesion-molecule expression in human umbilical vein endothelial cells (HUVECs). In estrogen-treated HUVECs, the mRNA and protein expression levels of adhesion molecules were quantified by real-time polymerase chain reaction and enzyme immunoassay. Additionally, a flow-chamber system was used to assess the effects of estrogens on the adherence of U937 monocytoid cells to HUVECs. Equilin, but not 17β-estradiol (E2) or other types of estrogen, significantly increased the mRNA (P < 0.01) and protein (P < 0.05) expression of the adhesion molecules E-selectin and intercellular adhesion molecule-1 as compared with levels in controls. Equilin treatment increased the adherence of U937 monocytoid cells to HUVECs relative to the that in the control (P < 0.05), decreased estrogen receptor (ER)β expression, and increased the expression of proteins involved in nuclear factor kappa-B (NF-κB) activation relative to levels in controls. Furthermore, the accumulation of NF-κB subunit p65 in HUVEC nuclei was promoted by equilin treatment. By contrast, E2 treatment neither increased the number of adhered monocytoid cells to HUVECs nor altered the expression of ERβ or NF-κB-activating proteins. Our findings suggest that in terms of the adhesion of monocytes at the onset of atherosclerosis, E2 may be preferable for estrogen-replacement therapy. Further studies comparing equilin treatment with that of E2 are needed to investigate their differential impacts on atherosclerosis.

Werner syndrome (WRN) DNA helicase and base excision repair (BER) factors maintain endothelial homeostasis

The accelerated ageing disease Werner Syndrome (WRN) is characterized by pronounced atherosclerosis. Here, we investigated the influence of WRN downregulation on the functionality of non-replicating human endothelial cells. RNAi-mediated downregulation of WRN reduces cell motility and enhances the expression of factors regulating adhesion, inflammation, hemostasis and vasomotor tone. Moreover, WRN influences endothelial barrier function and Ca2+-release, while cell adhesion, Dil-acLDL-uptake and the mRNA expression of NO-synthases (eNOS, iNOS) remained unaffected. Regarding motility, we propose that WRN affects Rac1/FAK/ß1-integrin-related mechanisms regulating cell polarity and directed motility. Since oxidative DNA base damage contributes to aging and atherosclerosis and WRN affects DNA repair, we investigated whether downregulation of base excision repair (BER) factors mimics the effects of WRN knock-down. Indeed, downregulation of particular WRN-interacting base excision repair (BER) proteins (APE1, NEIL1, PARP1) imitates the inhibitory effect of WRN on motility. Knock-down of OGG1, which does not interact with WRN, does not influence motility but increases the mRNA expression of E-selectin, ICAM, VCAM, CCL2 and VEGFR and stimulates adhesion. Thus, individual BER factors themselves differently impact endothelial cell functionality and homeostasis. Impairment of endothelial activities caused by genotoxic stressor (tBHQ) remained largely unaffected by WRN. Summarizing, both WRN, WRN-associated BER proteins and OGG1 promote the maintenance of endothelial cell homeostasis, thereby counteracting the development of ageing-related endothelial malfunction in non-proliferating endothelial cells.

Angiotensin II promotes pulmonary metastasis of melanoma through the activation of adhesion molecules in vascular endothelial cells

Hypertension is considered as one of the cancer progressive factors, and often found comorbidity in cancer patients. Renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure, and angiotensin II (Ang II) is well known pressor peptide associated with RAS. Ang II has been reported to accelerate progression and metastasis of cancer cells. However, its precise mechanisms have not been fully understood. In this study, we sought to elucidate the mechanisms by which Ang II exacerbates hematogenous metastasis in mouse melanoma cells, focusing the adhesion pathway in vascular endothelial cells. For this purpose, B16/F10 mouse melanoma cells, which do not express the Ang II type 1 receptor (AT1R), were intravenously injected into C57BL/6 mice. Two weeks after cell injection, the number of lung metastatic colonies was significantly higher in the Ang II-treated group (1 μg/kg/min) than in the vehicle-treated group. The AT1R blocker valsartan (40 mg/kg/day), but not the calcium channel blocker amlodipine (5 or 10 mg/kg/day), significantly suppressed the effect of Ang II. In endothelium-specific Agtr1a knockout mice, Ang II-mediated acceleration of lung metastases of melanoma cells was significantly diminished. Ang II treatment significantly increased E-selectin mRNA expression in vascular endothelial cells collected from lung tissues, and thus promoted adherence of melanoma cells to the vascular endothelium. Ang II-accelerated lung metastases of melanoma cells were also suppressed by treatment with anti-E-selectin antibody (20 mg/kg). Taken together, Ang II-treatment exacerbates hematogenous cancer metastasis by promoting E-selectin-mediated adhesion of cancer cells to vascular endothelial cells.

M1 macrophages are involved in folliculogenesis and M2 macrophages play an essential role for successful implantation

Early administration of low dose acetyl salicylic acid (Aspirin) in high risk women reduces the risk of early onset preeclampsia. This study aims to investigate the effect of aspirin on trophoblast integration and the its effect on angiogenic and invasive pathways in an in-vitro model of preeclampsia. Red fluorescent-labeled human uterine myometrial microvascular endothelial cells (UtMVECs) were seeded on matrigel to form endothelial networks. Green fluorescent-labeled trophoblastic HTR-8/SVneo cells were co-cultured with the endothelial networks with/without TNF-a (0.5 ng/mL) and/or aspirin (0.1 mM) for 24 h. Fluorescent images were captured and quantified by Image J to examine the effects of TNF-a and aspirin on the trophoblast-endothelial integration. Conditioned media were collected to measure free VEGF, PlGF and sFlt-1 by ELISA and PGF1a by Enzyme immunoassay (EIA). Cells were retrieved to examine mRNA expression of angiogenic factors (VEGF, PlGF and sFlt-1), invasion markers (MMP-2 and TIMP-1), endothelial cell activation markers (E-selectin and VCAM), eNOS and cyclooxygenase (COX)-2 by quantitative PCR. Aspirin reversed the inhibitory effect of TNF-a on trophoblast cell integration into endothelial cellular networks. TNF-a increased PGF1a production (128 ± 11%, p < 0.05), whilst aspirin reversed the TNF-a effect on PGF1a production (19 ± 4%, p < 0.01). TNF-a decreased the mRNA expression of PlGF, eNOS, MMP-2 and TIMP-1, and stimulated COX2, E-selectin and VCAM mRNA expression. Aspirin did not reverse the TNF-a effect on these molecules. Aspirin improves trophoblast cell integration into endothelial cellular networks by inhibiting the effect of TNF-a via PGI2 with no significant effect on antiangiogenic, invasive or endothelial activation markers.

The effect of acetyl salicylic acid (Aspirin) on trophoblast-endothelial interaction in vitro

Early administration of low dose acetyl salicylic acid (Aspirin) in high risk women reduces the risk of early onset preeclampsia. This study aims to investigate the effect of aspirin on trophoblast integration and the its effect on angiogenic and invasive pathways in an in-vitro model of preeclampsia. Red fluorescent-labeled human uterine myometrial microvascular endothelial cells (UtMVECs) were seeded on matrigel to form endothelial networks. Green fluorescent-labeled trophoblastic HTR-8/SVneo cells were co-cultured with the endothelial networks with/without TNF-a (0.5ng/mL) and/or aspirin (0.1mM) for 24h. Fluorescent images were captured and quantified by Image J to examine the effects of TNF-a and aspirin on the trophoblast-endothelial integration. Conditioned media were collected to measure free VEGF, PlGF and sFlt-1 by ELISA and PGF1a by Enzyme immunoassay (EIA). Cells were retrieved to examine mRNA expression of angiogenic factors (VEGF, PlGF and sFlt-1), invasion markers (MMP-2 and TIMP-1), endothelial cell activation markers (E-selectin and VCAM), eNOS and cyclooxygenase (COX)-2 by quantitative PCR. Aspirin reversed the inhibitory effect of TNF-a on trophoblast cell integration into endothelial cellular networks. TNF-a increased PGF1a production (128±11%, p<0.05), whilst aspirin reversed the TNF-a effect on PGF1a production (19±4%, p<0.01). TNF-a decreased the mRNA expression of PlGF, eNOS, MMP-2 and TIMP-1, and stimulated COX2, E-selectin and VCAM mRNA expression. Aspirin did not reverse the TNF-a effect on these molecules. Aspirin improves trophoblast cell integration into endothelial cellular networks by inhibiting the effect of TNF-a via PGI2 with no significant effect on antiangiogenic, invasive or endothelial activation markers.

Aspergillus fumigatus-induced early inflammatory response in pulmonary microvascular endothelial cells: Role of p38 MAPK and inhibition by silibinin

Human invasive pulmonary aspergillosis (IPA) is a serious infectious disease mainly caused by Aspergillus fumigatus (A. fumigatus). Pulmonary microvascular endothelial cells (PMVECs) are important ones in the human lung tissue. However, it remains unclear about the role of PMVECs in IPA. In the present study, we cocultured PMVECs with A. fumigatus. We observed that A. fumigatus induced dose- and time-dependent increases of interleukin 6 (IL-6), interleukin 1β (IL-1β) and intercellular adhesion molecule 1 (ICAM-1) concentration in the cultures. Significant increases in IL-6, IL-1β, E-selectin, and ICAM-1 mRNA expression were also observed in the cultures treated with A. fumigatus. While preincubation with SB203580 (10μM) did not cause significant changes in IL-6, IL-1β and ICAM-1 concentration in the cocultures, significant IL-6, IL-1β and ICAM-1 concentration decreases were observed in the cocultures preincubated with SB203580 (20μM). Neither SP600125 (10-20μM) nor PD98059 (10-20μM) caused significant changes in IL-6, IL-1β and ICAM-1 concentration in the cocultures. PCR results also showed that SB203580 (20μM) (neither SP600125 (20μM) nor PD98059 (20μM)) preincubation significantly decreased IL-6, IL-1β, E-selectin and ICAM-1 mRNA expression in the cocultures. In addition, significant p38 MAPK phosphorylation increase was observed in the PMVECs cultures treated with A. fumigatus. Furthermore, silibinin pre-treatment and post-treatment were observed to significantly down-regulate mRNA and protein expression of proinflammatory factors and adhesion molecules in the cocultures. Finally, we observed that silibinin significantly inhibited A. fumigatus-induced p38 MAPK activation in PMVECs. Our results indicated that PMVECs might participate in IPA early inflammation which is mediated by p38 MAPK. Silibinin may inhibit A. fumigatus-induced inflammation in PMVECs through p38 MAPK.

Serum of patients with antiphospholipid syndrome induces adhesion molecules in endothelial cells

ObjectiveThe antiphospholipid syndrome (APS) is a systemic auto-immune disease with an unclear pathophysiology. The aim of our study was to understand the development of APS on a cellular level. Therefore, we analyzed the influence of human serum of APS patients on endothelial expression of specific genes and proteins in comparison to a control group. MethodsIn this study, we analyzed the expression of ICAM-1, VCAM-1, E-selectin and annexin V in primary cultures of human umbilical vein endothelial cells (HUVEC) in response to 10% (v/v) serum of control patients (n = 6), patients with systemic lupus erythematosus (SLE) and no APS (n = 4) or APS patients (n = 9) for 24 h. Total RNA was prepared from confluent endothelial cell layers and mRNA expression of ICAM-1, VCAM-1 and E-selectin was analyzed by reverse transcription polymerase-chain reaction (RT-PCR). The protein expression was determined by Western blot. Serum protein concentrations of soluble forms of adhesion molecules sICAM-1 and sVCAM-1 were quantified by ELISA. Gene expression data were correlated with clinical parameters. ResultsThe mRNA expression of ICAM-1 was increased in cells incubated with serum from APS patients (166 ± 22% of control; P = 0.023). Serum of patients with (SLE)/no APS caused a 1.4-fold higher ICAM-1 mRNA level. Western blot analysis showed an increase in protein expression of adhesion molecules ICAM-1 (260 ± 49%; P = 0.011) and VCAM-1 (357 ± 97%; P = 0.023) in cells that were incubated with serum from APS patients. Plasma analysis showed elevated levels of sVCAM-1 in APS patients (189 ± 34%; P = 0.045) compared to the levels measured in the control group. The sVCAM-1 plasma level was correlating with the frequency of abortions. ConclusionAn augmented expression of endothelial adhesion molecules is involved in the pathophysiology of patients with antiphospholipid syndrome.

Therapeutic effects of rosmarinic acid on airway responses in a murine model of asthma

Rosmarinic acid (RA) is an active component of a traditional Chinese herbal medicine. Previously, we reported that RA exerted a strong anti-inflammatory effect in a mouse acute lung injury model. Therefore, we hypothesized that RA might also have potential therapeutic effects in a murine model of asthma. In this study, we aimed to evaluate the anti-asthmatic activity of RA and explored its possible molecular mechanisms of action. Female BALB/c mice that had been sensitized to and challenged with ovalbumin (Ova) were treated with RA (20mg/kg) 1h after challenge. The results showed that RA greatly diminished the number of inflammatory cells and the production of Th2 cytokines in the bronchoalveolar lavage fluid (BALF); significantly reduced the secretion of total IgE, Ova-specific IgE, and eotaxin; and markedly ameliorated airway hyperresponsiveness (AHR) compared with Ova-induced mice. Histological studies further revealed that RA substantially decreased inflammatory cells infiltration and mucus hypersecretion compared with Ova-induced mice. Moreover, our results suggested that the protective effects of RA were mediated by the inhibition of JNK and p38 MAPK phosphorylation and nuclear factor-κB (NF-κB) activation. Furthermore, RA treatment resulted in a significant reduction in the mRNA expression of AMCase, CCL11, CCR3, Ym2 and E-selectin in lung tissue. These findings suggest that RA may effectively delay the development of airway inflammation and could thus be used as a therapy for allergic asthma.

MTUS1 silencing promotes E-selectin production through p38 MAPK-dependent CREB ubiquitination in endothelial cells

BackgroundEndothelial cell activation is thought to be a key event in atherosclerosis. p38 mitogen-activated protein kinase (p38 MAPK) plays an important role in regulating pro-inflammatory cytokine production in endothelial cells (ECs), however, how p38 MAPK is controlled in EC activation remain unclear. In this study, we investigated the effect of mitochondrial tumor suppressor 1 (MTUS1) on p38 MAPK activation, cytokine induction and the underlying molecular mechanisms in ECs. Methods and resultsUsing qPCR and ELISA methods, we found that knockdown of MTUS1 led to a marked increase in the mRNA and protein expression of E-selectin (SELE) and monocyte chemotactic protein-1 in ECs, which is accompanied with increased phosphorylation of p38 MAPK (Thr180/Tyr182), MKK3/6 (Ser 189) and IκBα (Ser 32). Using luciferase reporter assay, we found that MTUS1 silencing also activated NF-κB transcriptional activity. The inhibition of p38 MAPK and NF-κB pathway was shown to abrogate MTUS1 silencing-induced cytokine expression in ECs. Furthermore, MTUS1 silencing induced p38 MAPK-dependent ubiquitination of cAMP-response element binding protein (CREB) which potentiated CREB-binding protein-mediated NF-κB p65 acetylation and binding to the promoter of the SELE gene. Conversely, adenovirus-mediated overexpression of MTUS1 inhibited p38 MAPK activation in ECs in vitro and in vivo. Importantly, decreased expression of MTUS1 and CREB, accompanied with induced activation of p38 MAPK were observed in aortas of apoE−/− mice after high-fat diet challenge. ConclusionsOur findings showed that MTUS1 regulates the p38 MAPK-mediated cytokine production in ECs. MTUS1 gene probably plays a protective role against pro-inflammatory response of ECs.

Nanoformulation of curcumin protects HUVEC endothelial cells against ionizing radiation and suppresses their adhesion to monocytes: potential in prevention of radiation-induced atherosclerosis.

To investigated the potential of a novel dendrosomal nanoformulation of curcumin (DNC) in blocking radiation-induced changes in irradiated human umbilical vein endothelial cells (HUVECs), and their adhesion to human THP-1 monocytoid cells. Co60 gamma rays reduced viability, raised the expression of adhesion molecules, ICAM-1, VCAM-1 and E-selectin (mRNA and protein), augmented the adhesion of THP-1 cells to HUVECs, activated NF-κB binding, increased the release of pro-inflammatory cytokines (IL-6, IL-8 and MCP-1) and induced oxidative damage (reduced glutathione declined, while 8-OHdG and TBARS increased). 5µM DNC significantly inhibited these radiation-induced changes, activated the Nrf-2 pathway, and effectively suppressed THP-1 adhesion to HUVECs, implicating p38 MAPK signaling. DNC treatment is a potential preventive method against inflammation and vascular damage from ionizing radiation.