Anti-Inflammatory Activity of Ferula assafoetida Oleo-Gum-Resin (Asafoetida) against TNF-α-Stimulated Human Umbilical Vein Endothelial Cells (HUVECs).

Inflammation is the body's biological reaction to endogenous and exogenous stimuli. Recent studies have demonstrated several anti-inflammatory properties of Ferula species. In this paper, we decided to study the anti-inflammatory effect of ethanolic extract of Ferula assafoetida oleo-gum-resin (asafoetida) against TNF-α-stimulated human umbilical vein endothelial cells (HUVECs). HUVECs were cultured in a flat-bottom plate and then treated with ethanolic extract of asafoetida (EEA, 0-500 µg/ml) and TNF-α (0-100 ng/ml) for 24 h. We used the MTT test to assess cell survival. In addition, the LC-MS analysis was performed to determine the active substances. HUVECs were pretreated with EEA and then induced by TNF-α. Intracellular reactive oxygen species (ROS) and adhesion of peripheral blood mononuclear cells (PBMCs) to HUVECs were evaluated with DCFH-DA and CFSE fluorescent probes, respectively. Gene expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin and surface expression of ICAM-1 protein were measured using real-time PCR and flow cytometry methods, respectively. While TNF-α significantly increased intracellular ROS formation and PBMC adhesion to TNF-α-induced HUVECs, the pretreatment of HUVECs with EEA (125 and 250 µg/ml) significantly reduced the parameters. In addition, EEA pretreatment decreased TNF-α-induced mRNA expression of VCAM-1 and surface protein expression of ICAM-1 in the target cells. Taken together, the results indicated that EEA prevented ROS generation, triggered by TNF-α, and inhibited the expression of VCAM-1 and ICAM-1, leading to reduced PBMC adhesion. These findings suggest that EEA can probably have anti-inflammatory properties.

ESDN inhibits melanoma progression by blocking E-selectin expression in endothelial cells via STAT3.

An interactive crosstalk between tumor and stroma cells is essential for metastatic melanoma progression. We evidenced that ESDN/DCBLD2/CLCP1 plays a crucial role in endothelial cells during the spread of melanoma. Precisely, increased extravasation and metastasis formation were revealed in ESDN-null mice injected with melanoma cells, even if the primary tumor growth, vessel permeability, and angiogenesis were not enhanced. Interestingly, improved adhesion of melanoma cells to ESDN-depleted endothelial cells was observed, due to the presence of higher levels of E-selectin transcripts/proteins in ESDN-defective cells. In accordance with these results, anticorrelation was observed between ESDN and E-selectin in human endothelial cells. Most importantly, our data revealed that cimetidine, an E-selectin inhibitor, was able to block cell adhesion, extravasation, and metastasis formation in ESDN-null mice, underlying a major role of ESDN in E-selectin transcription upregulation, which according to our data, may presumably be linked to STAT3. Based on our results, we propose a protective role for ESDN during the spread of melanoma and reveal its therapeutic potential.

Expression of functional E-selectin ligands on the plasma membrane of carcinoma cells correlates with poor prognosis in clear cell renal cell carcinoma.

OBJECTIVES: Given the relatively high frequency of metastatic recurrence of clear cell renal cell carcinoma (ccRCC), reliable prognostic markers of ccRCC, particularly those associated with metastasis, are needed. Here, in search of those factors, we assessed the contribution of sialyl Lewis x (sLex) and sialyl Lewis a (sLea), as well as functional E-selectin ligand carbohydrates expressed on carcinoma cells, to metastasis and consequent poor prognosis in ccRCC. MATERIALS AND METHODS: Patients who underwent surgical resection (curative nephrectomy) for RCC, and whose post-operative pathological diagnosis was ccRCC (n = 117) were enrolled in this study. Expression of sLex/sLea carbohydrate antigens in ccRCC was evaluated by immunohistochemistry with an anti-sLex/sLea monoclonal antibody HECA-452. To evaluate membrane expression of sLex/sLea carbohydrate antigens quantitatively, we employed a histological scoring system used to evaluate membrane expression of human epidermal growth factor receptor 2 (HER2) in breast cancer. We also conducted an E-selectin•IgM chimera in situ binding assay to assess expression of functional E-selectin ligand carbohydrates in ccRCC. We then carried out statistical analysis to determine whether membrane expression of HECA-452-reactive sLex/sLea glycans as well as of E-selectin•IgM-binding functional E-selectin ligand carbohydrates correlates with progression-free, overall, or cancer-specific survival. RESULTS: Based on HECA-452 immunochemistry, 106 of 117 ccRCC specimens expressed detectable levels of sLex/sLea glycans, primarily on the plasma membrane, and of those, 31 that showed robust membrane expression were judged as HECA-452-positive. Membrane expression of HECA-452-positive sLex/sLea glycans correlated with shortened progression-free and overall survival. Moreover, in in situ analysis, these HECA-452-positive ccRCC tissues were decorated with E-selectin•IgM chimeric proteins, calcium-dependently. Comparable analysis in normal kidney showed both HECA-452 positivity and chimera binding almost exclusively in epithelial cells that constitute proximal tubules. Membrane expression of functional E-selectin ligand carbohydrates, as detected by the E-selectin•IgM chimera, correlated more significantly with poor prognosis of patients, namely, shortened progression-free, overall and cancer-specific survival, than did HECA-452 positivity. CONCLUSIONS: Expression of E-selectin•IgM-binding functional E-selectin ligand carbohydrates can serve as a reliable and potentially superior prognostic biomarker of patients with ccRCC.

Peucedanum ostruthium Inhibits E-Selectin and VCAM-1 Expression in Endothelial Cells through Interference with NF-κB Signaling.

Twenty natural remedies traditionally used against different inflammatory diseases were probed for their potential to suppress the expression of the inflammatory markers E-selectin and VCAM-1 in a model system of IL-1 stimulated human umbilical vein endothelial cells (HUVEC). One third of the tested extracts showed in vitro inhibitory effects comparable to the positive control oxozeaenol, an inhibitor of TAK1. Among them, the extract derived from the roots and rhizomes of Peucedanum ostruthium (i.e., Radix Imperatoriae), also known as masterwort, showed a pronounced and dose-dependent inhibitory effect. Reporter gene analysis demonstrated that inhibition takes place on the transcriptional level and involves the transcription factor NF-κB. A more detailed analysis revealed that the P. ostruthium extract (PO) affected the phosphorylation, degradation, and resynthesis of IκBα, the activation of IKKs, and the nuclear translocation of the NF-κB subunit RelA. Strikingly, early effects on this pathway were less affected as compared to later ones, suggesting that PO may act on mechanism(s) that are downstream of nuclear translocation. As the majority of cognate NF-κB inhibitors affect upstream events such as IKK2, these findings could indicate the existence of targetable signaling events at later stages of NF-κB activation.

Effect of Tribulus Terrestris L. on Expression of ICAM-1, VCAM-1, E-Selectin and Proteome Profile of Human Endothelial Cells In-Vitro.

BACKGROUND: Atherosclerosis is a chronic inflammation that interferes with blood arteries functions due to the accumulation of low density lipids and cholesterol. OBJECTIVE: To investigate the effect of aqueous extract and saponin fraction of Tribulus terrestris L. (TT) on the proteome and expression of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in the human umbilical vein endothelial cell (HUVEC) and human bone marrow endothelial cell (HBMEC) lines. METHODS: Two cell lines were cultured and induced with lipopolysaccharide (LPS). The primed cells were then treated with aqueous extract and saponin fraction of TT. The protein profile of the endothelial cells was assessed under normal and LPS-induced conditions using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 2D gel electrophoresis (2-DE). The levels of VCAM-1, ICAM-1, and E-selectin were estimated by use of western blotting. RESULTS: LPS-induced HUVECs and HBMECs were shown to significantly increase the expression of ICAM-1, VCAM-1, and E-selectin in comparison to control groups. Our findings revealed that TT extract resulted in significantly more reduced levels of proteome (80 spots) as well as all the three mentioned proteins compared with the effect of saponin fraction alone. CONCLUSION: TT extract and its saponin fraction exerted anti-inflammatory effects on HUVEC and HBMEC lines and reduced the expression of ICAM-1, VCAM-1, and E-selectin. However, the anti-inflammatory effect of aqueous extract was greater than that of saponin fraction. Therefore, TT could be considered as a potential candidate for the treatment or prevention of atherosclerosis.

New A-homo lactam D-homo lactone androstane derivative: Synthesis and evaluation of cytotoxic and anti-inflammatory activities in vitro.

This paper describes the synthesis of a new A-homo lactam D-homo lactone androstane derivative from dehydroepiandrosterone. To evaluate the impact of the introduction of nitrogen in the parental scaffold on biological activity, a new androstane enamide-type lactam derivative was prepared and characterized. The new compound as well as starting compounds were screened for cytotoxic, anti-angiogenic and anti-inflammatory activities using several human cancer cell lines (MCF-7, MDA-MB-231, PC3, CEM, G-361, HeLa), endothelial (HUVEC) and non-tumour (MRC-5 and BJ) cell lines. Strong cytotoxic and anti-inflammatory activity with a broad therapeutical window was demonstrated by the A-homo lactam D-homo lactone androstane derivative. The induction of apoptosis in treated PC3 cultures was confirmed using apoptotic morphology screening and a fluorescent double-staining method. New A-homo lactam D-homo lactone androstane derivative induced apoptosis more than the tested reference compounds, Formestane and Doxorubicin. An in silico ADME analysis showed that the compounds possess drug-like properties.

Cenicriviroc inhibits trans-endothelial passage of monocytes and is associated with impaired E-selectin expression.

Incidences of cardiovascular diseases (CVD) are high among virologically suppressed HIV-infected individuals. Monocyte activation and trafficking are key mechanisms in the evolution of CVD. We studied the ability of cenicriviroc (CVC), a dual C-C chemokine receptor type 2 (CCR2) and CCR5 antagonist, to influence the migration of monocytes from HIV-infected individuals on antiretroviral therapy (ART). Monocytes were derived from 23 ART-suppressed HIV-infected and 16 HIV-uninfected donors. In a trans-endothelial migration model, monocytes, and human aortic endothelial cells (HAoECs) were exposed to cenicriviroc and migrated monocytes, quantified. Expression of CCR2 and CCR5 on monocytes and adhesion molecules (E-selectin, ICAM-1, VCAM-1, PECAM-1, and CD99) on HAoECs were measured. The single antagonists, BMS-22 (CCR2), and maraviroc (CCR5), served as controls. When both HAoECs and monocytes together were exposed to the antagonists, cenicriviroc led to a greater decrease in monocyte migration compared to BMS-22 or vehicle in both HIV-infected and HIV-uninfected groups (P < 0.05), with maraviroc having no inhibitory effect. Cenicriviroc treatment of HAoECs alone decreased monocyte migration in the HIV-infected group when compared to vehicle (P < 0.01). Inhibition of migration was not evident when monocytes alone were exposed to cenicriviroc, BMS-22 or maraviroc. Incubation of HAoECs with cenicriviroc decreased E-selectin expression (P = 0.045) but had limited effects on the other adhesion molecules. Cenicriviroc inhibits monocyte trans-endothelial migration more effectively than single chemokine receptor blockade, which may be mediated via disruption of monocyte-endothelial tethering through reduced E-selectin expression. Cenicriviroc should be considered as a therapeutic intervention to reduce detrimental monocyte trafficking.

ß-Caryophyllene protects against alcoholic steatohepatitis by attenuating inflammation and metabolic dysregulation in mice.

BACKGROUND AND AIMS: ß-Caryophyllene (BCP) is a plant-derived FDA approved food additive with anti-inflammatory properties. Some of its beneficial effects in vivo are reported to involve activation of cannabinoid CB2 receptors that are predominantly expressed in immune cells. Here, we evaluated the translational potential of BCP using a well-established model of chronic and binge alcohol-induced liver injury. METHODS: In this study, we investigated the effects of BCP on liver injury induced by chronic plus binge alcohol feeding in mice in vivo by using biochemical assays, real-time PCR and histology analyses. Serum and hepatic BCP levels were also determined by GC/MS. RESULTS: Chronic treatment with BCP alleviated the chronic and binge alcohol-induced liver injury and inflammation by attenuating the pro-inflammatory phenotypic `M1` switch of Kupffer cells and by decreasing the expression of vascular adhesion molecules intercellular adhesion molecule 1, E-Selectin and P-Selectin, as well as the neutrophil infiltration. It also beneficially influenced hepatic metabolic dysregulation (steatosis, protein hyperacetylation and PPAR-α signalling). These protective effects of BCP against alcohol-induced liver injury were attenuated in CB2 receptor knockout mice, indicating that the beneficial effects of this natural product in liver injury involve activation of these receptors. Following acute or chronic administration, BCP was detectable both in the serum and liver tissue homogenates but not in the brain. CONCLUSIONS: Given the safety of BCP in humans, this food additive has a high translational potential in treating or preventing hepatic injury associated with oxidative stress, inflammation and steatosis. LINKED ARTICLES: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

NFκB transcription factor (p65) immunohistochemistry in leprosy dermal microvasculature.

Leprosy caused by Mycobacterium leprae is characterized by a spectrum of clinical manifestations that are determined by the predominant immunological profile of the host. The recruitment of leukocytes to the sites of injury can influence the development of these profiles. Cell adhesion molecules such as ICAM-1, VCAM-1 and CD62E participate in this process and their expression is regulated by transcriptions factors such as NFκB. To correlate the expression of cell adhesion molecules and NFκB (p65) in leprosy lesions, 30 skin biopsies of patients with leprosy [16 with the tuberculoid (TT) or borderline tuberculoid (BT) forms and 14 with the lepromatous (LL) or borderline lepromatous (BL) forms] were analyzed by immunohistochemistry. A larger mean number of cells expressing VCAM-1 (BT/TT: 18.28 ± 1.4; BL/LL: 10.67 ± 1.2; p = 0.0002), ICAM-1 (BT/TT: 9.92 ± 1.1; BL/LL: 5.87 ± 1.0; p = 0.0084) and CD62E (BT/TT: 13.0 ± 1.5; BL/LL: 2.58 ± 0.3; p = 0.0001) were observed in BT and TT lesions. The mean number of cells expressing NFκB was similar in the two clinical forms (BT/TT: 2.21 ± 2.7; BL/LL: 2.35 ± 3.1;p = 0.9285). No significant correlation was observed between expression of the transcription factor and adhesion molecules analyzed. The synthesis of ICAM-1, VCAM-1 and CD62E depends on the activation of NFκB, which acts synergistically with other transcription factors. Adequate activation of intracellular signaling pathways results in the production of endothelial adhesion molecules, contributing to the recruitment of cells to the site of injury and thus eliciting an effective inflammatory response in the elimination of the bacillus.

Does one size fit all? The role of body mass index and waist circumference in systemic inflammation in midlife by race and gender.

OBJECTIVE: This study investigates the associations of body mass index (BMI) and waist circumference (WC) with markers of systemic inflammation in midlife by race and gender. DESIGN: Data were obtained from the Survey of Midlife in the United States, a cross-sectional, observational study of Americans 35 years old or older (White men: N = 410; White women: N = 490; Black men: N = 58; Black women: N = 117). Inflammation was measured by concentrations of fibrinogen and C-reactive protein (CRP) in fasting plasma and concentrations of E-selectin and interleukin-6 (IL-6) in fasting serum. Anthropometric data were used to obtain BMI and WC. Socio-demographic and health-related factors were assessed with a survey. Multivariate models by race and gender were estimated to test the roles of BMI and WC for each inflammation marker. RESULTS: Compared to White men, Black women have higher BMI and higher levels of all four inflammation markers; White women have lower BMI, lower WC, and lower E-selectin and fibrinogen but higher CRP; and Black men have higher fibrinogen. After adjusting for socio-demographic and health-related covariates as well as perceived discrimination, WC is associated with all four markers of inflammation among White men and women; with three markers (fibrinogen, CRP, and IL-6) of inflammation among Black women; and with CRP (and marginally with fibrinogen and E-selectin) among Black men. BMI is associated with higher CRP and fibrinogen among Black men (marginally so for White men) but not for women of either race. CONCLUSIONS: WC shows more consistent associations with inflammation markers than BMI, although the relationships vary by inflammation marker and population group. Our findings suggest that WC is a risk factor for systemic inflammation among White and Black men and women, and BMI is an additional risk factor for Black men.

Recruited monocytic myeloid-derived suppressor cells promote the arrest of tumor cells in the premetastatic niche through an IL-1ß-mediated increase in E-selectin expression.

The tumor premetastatic niche initiated by primary tumors is constructed by multiple molecular factors and cellular components and provides permissive condition that allows circulating tumor cells to successfully metastasize. Myeloid-derived suppressor cells (MDSCs), a population of immature cells in pathological conditions, play a critical role in the formation of the premetastatic niche. However, few researches are focused on the function of monocytic MDSCs (mo-MDSCs), a subtype of MDSCs, in the construction of the niche. Here, we show that the number of mo-MDSCs is significantly increased in the premetastatic lungs of tumor-bearing mice, thus promoting tumor cell arrest and metastasis. Before the arrival of tumor cells, the lung-recruited mo-MDSCs produced IL-1ß, thereby increasing E-selectin expression and promoting tumor cell arrest on endothelial cells. Depletion of mo-MDSCs in the premetastatic lungs decreased IL-1ß production, resulting in reduced E-selectin expression. In addition, compared with alveolar macrophages and interstitial macrophages, mo-MDSCs were the major source of IL-1ß expression in the premetastatic lungs. Cytokine array analyses and transwell experiments revealed that CCL12 recruits mo-MDSCs to premetastatic lungs. CCL12 knockdown in tumor-bearing mice significantly decreased mo-MDSC infiltration into the premetastatic lungs, leading to reduced E-selectin expression. Overall, the permissive conditions produced by the infiltrated mo-MDSCs correlated with increased tumor cell arrest and metastasis. These results reveal a novel role of mo-MDSCs in constructing the premetastatic niche. Thus, inhibition of mo-MDSCs infiltration may change the premetastatic niche to normal condition and attenuate tumor metastasis.

Prenylated Polyphenols from Clusiaceae and Calophyllaceae with Immunomodulatory Activity on Endothelial Cells.

Endothelial cells (ECs) are key players in inflammation and immune responses involved in numerous pathologies. Although attempts were experimentally undertaken to prevent and control EC activation, drug leads and probes still remain necessary. Natural products (NPs) from Clusiaceous and Calophyllaceous plants were previously reported as potential candidates to prevent endothelial dysfunction. The present study aimed to identify more precisely the molecular scaffolds that could limit EC activation. Here, 13 polyphenols belonging to 5 different chemical types of secondary metabolites (i.e., mammea coumarins, a biflavonoid, a pyranochromanone acid, a polyprenylated polycyclic acylphloroglucinol (PPAP) and two xanthones) were tested on resting and cytokine-activated EC cultures. Quantitative and qualitative changes in the expression of both adhesion molecules (VCAM-1, ICAM-1, E-selectin) and major histocompatibility complex (MHC) molecules have been used to measure their pharmaceutical potential. As a result, we identified 3 mammea coumarins that efficiently reduce (up to >90% at 10 µM) both basal and cytokine-regulated levels of MHC class I, class II, MICA and HLA-E on EC surface. They also prevented VCAM-1 induction upon inflammation. From a structural point of view, our results associate the loss of the free prenyl group substituting mammea coumarins with a reduced cellular cytotoxicity but also an abrogation of their anti-inflammatory potential and a reduction of their immunosuppressive effects. A PPAP, guttiferone J, also triggers a strong immunomodulation but restricted to HLA-E and MHC class II molecules. In conclusion, mammea coumarins with a free prenyl group and the PPAP guttiferone J emerge as NPs able to drastically decrease both VCAM-1 and a set of MHC molecules and to potentially reduce the immunogenicity of the endothelium.

Characterization of Innate Immune Responses of Human Endothelial Cells Induced by Porphyromonas gingivalis and Their Derived Outer Membrane Vesicles.

Atherosclerosis, a chronic inflammatory disease of the blood vessels, is one of the most common causes of morbidity and mortality world-wide. Involvement of Porphyromonas gingivalis in atherosclerosis is supported by observations from epidemiological, clinical, immunological, and molecular studies. Previously we reported that P. gingivalis vesicles have a much higher invasive efficiency than their originating cells. Here, we further compare the role of P. gingivalis cells and their vesicles in expression of chemoattractant proteins including CXCL1, CXCL2, and CXCL8, and adhesive molecules such as E-selectin in human umbilical vein endothelial cells (HUVECs). Both P. gingivalis 33277 cells and vesicles were able to up-regulate expression of these molecules, while the vesicles acted as more potent inducers of the inflammatory response associated with the development of atherosclerosis, consequently resulting in significant monocyte adhesion to a monolayer of HUVECs. Interestingly, we found that elevated expression of CXCL8 and E-selectin in endothelial cells induced by P. gingivalis correlated with the invasive ability of P. gingivalis cells and vesicles. Non-invasive bacterial cells and vesicles had no effect on expression of these genes. This study highlights the potential risk of P. gingivalis cells and vesicles in initiation of atherosclerosis and provides a potential target for the development of novel therapeutics against bacteria-associated atherosclerosis.

Bleomycin increases neutrophil adhesion to human vascular endothelial cells independently of upregulation of ICAM-1 and E-selectin.

AIM OF THE STUDY: Bleomycin-induced lung disease is a serious complication of therapy characterized by alveolar injury, cytokine release, inflammatory cell recruitment, and eventually pulmonary fibrosis. The mechanisms underlying bleomycin-induced pulmonary fibrosis may be relevant to other progressive scarring diseases of the lungs. Pulmonary vascular endothelial cells are critically involved in immune cell extravasation at sites of injury through adhesion molecule expression and cytokine release. We sought to determine the effects of bleomycin on adhesion molecule expression and cytokine release by pulmonary vascular endothelial cells, and their functional relevance to inflammatory cell recruitment. MATERIALS AND METHODS: The effects of pharmacologically relevant concentrations of bleomycin on adhesion molecule expression and cytokine release by human vascular endothelial cells in vitro were studied by flow cytometry, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. A flow chamber model was used to assess the functional consequences on adhesion of flowing human neutrophils to endothelial cell monolayers. RESULTS: Bleomycin increased intercellular adhesion molecule 1 (ICAM-1; CD54), vascular cell adhesion molecule (VCAM-1; CD106), and E-selectin (CD62E) expression, and increased monocyte chemoattractant protein (MCP-1) and interleukin (IL-8) release by endothelial cells. Increases in protein expression were accompanied by increased mRNA transcription. In contrast, there was no direct effect of bleomycin on the profibrotic cytokines transforming growth factor-beta (TGF-ß), platelet-derived growth factor-BB (PDGF-BB), or endothelin-1. Under flow conditions, endothelial cells exposed to bleomycin supported increased neutrophil adhesion which was independent of ICAM-1 or E-selectin. CONCLUSION: Our findings demonstrate that bleomycin promotes endothelial-mediated inflammation and neutrophil adhesion. These mechanisms may contribute to the development of pulmonary fibrosis by supporting immune cell recruitment in the lungs.

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

BACKGROUND: Endothelial 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 RESULTS: Using 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. CONCLUSIONS: Our 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.

Impact of Bi-Axial Shear on Atherogenic Gene Expression by Endothelial Cells.

This study demonstrated the effects of the directionality of oscillatory wall shear stress (WSS) on proliferation and proatherogenic gene expression (I-CAM, E-Selectin, and IL-6) in the presence of inflammatory mediators leukotriene B4 (LTB4) and bacterial lipopolysaccharide (LPS) from endothelial cells grown in an orbiting culture dish. Computational fluid dynamics (CFD) was applied to quantify the flow in the dish, while an analytical solution representing an extension of Stokes second problem was used for validation. Results indicated that WSS magnitude was relatively constant near the center of the dish and oscillated significantly (0-0.9 Pa) near the side walls. Experiments showed that LTB4 dominated the shear effects on cell proliferation and area. Addition of LPS didn't change proliferation, but significantly affected cell area. The expression of I-CAM1, E-Selectin and IL-6 were altered by directional oscillatory shear index (DOSI, a measure of the biaxiality of oscillatory shear), but not shear magnitude. The significance of DOSI was further reinforced by the strength of its interactions with other atherogenic factors. Hence, directionality of shear appears to be an important factor in regulating gene expression and provides a potential explanation of the propensity for increased vascular lesions in regions in the arteries with oscillating biaxial flow.

Two E-selectin ligands, BST-2 and LGALS3BP, predict metastasis and poor survival of ER-negative breast cancer.

Distant metastases account for the majority of cancer-related deaths in breast cancer. The rate and site of metastasis differ between estrogen receptor (ER)-negative and ER-positive tumours, and metastatic fate can be very diverse even within the ER-negative group. Characterisation of new pro-metastatic markers may help to identify patients with higher risk and improve their care accordingly. Selectin ligands aberrantly expressed by cancer cells promote metastasis by enabling interaction between circulating tumour cells and endothelial cells in distant organs. These ligands consist in carbohydrate molecules, such as sialyl-Lewis x antigen (sLex), borne by glycoproteins or glycolipids on the cancer cell surface. We have previously demonstrated that the molecular scaffold presenting sLex to selectins (e.g. glycolipid vs. glycoproteins) was crucial for these interactions to occur. Moreover, we reported that detection of sLex alone in breast carcinomas was only of limited prognostic value. However, since sLex was found to be carried by several glycoproteins in cancer cells, we hypothesized that the combination of the carbohydrate with its carriers could be more relevant than each marker independently. In this study, we addressed this question by analysing sLex expression together with two glycoproteins (BST-2 and LGALS3BP), shown to interact with E-selectin in a carbohydrate-dependent manner, in a cohort of 249 invasive breast cancers. We found both glycoproteins to be associated with distant metastasis risk and poorer survival. Importantly, concomitant high expression of BST-2 with sLex defined a sub-group of patients with ER-negative tumours displaying higher risks of liver and brain metastasis and a 3-fold decreased survival rate.

A novel microfluidic assay reveals a key role for protein kinase C δ in regulating human neutrophil-endothelium interaction.

A key step in neutrophil-mediated tissue damage is the migration of activated neutrophils across the vascular endothelium. Previously, we identified protein kinase C δ as a critical regulator of neutrophil migration in sepsis but did not identify specific steps in migration. In this study, we used our novel biomimetic microfluidic assay to delineate systematically the mechanism by which protein kinase C δ regulates individual steps in human neutrophil-endothelial interaction during inflammation. The biomimetic microfluidic assay includes a network of vascular channels, produced from in vivo images connected to a tissue compartment through a porous barrier. HUVECs cultured in vascular channels formed a complete lumen under physiologic shear flow. HUVECs were pretreated with TNF-α ± a protein kinase C δ inhibitor, and the tissue compartment was filled with a chemoattractant (fMLP or IL-8). Under physiologic shear flow, the role of protein kinase C δ on spatial and temporal neutrophil adherence/migration was quantified. Protein kinase C δ inhibition significantly reduced neutrophil adhesion in response to fMLP and IL-8 only under low shear rate and near bifurcations. Protein kinase C δ inhibition also decreased adherence to nonactivated HUVECs in response to fMLP or IL-8. Protein kinase C δ inhibition reduced neutrophil migration into the tissue compartment in response to fMLP and to a lesser degree, to IL-8. Antibody-coated microparticles demonstrated that protein kinase C δ inhibition down-regulated E-selectin and ICAM-1 but not VCAM-1 expression. With the use of a physiologically relevant in vitro model system, we demonstrate that protein kinase C δ plays an important role in the regulation of neutrophil adherence/migration during inflammation and identifies key steps regulated by protein kinase C δ in neutrophil-endothelial interactions.

Complement MASP-1 enhances adhesion between endothelial cells and neutrophils by up-regulating E-selectin expression.

The complement system and neutrophil granulocytes are indispensable in the immune response against extracellular pathogens such as bacteria and fungi. Endothelial cells also participate in antimicrobial immunity largely by regulating the homing of leukocytes through their cytokine production and their pattern of cell surface adhesion molecules. We have previously shown that mannan-binding lectin-associated serine protease-1 (MASP-1), a complement lectin pathway enzyme, is able to activate endothelial cells by cleaving protease activated receptors, which leads to cytokine production and enables neutrophil chemotaxis. Therefore, we aimed to investigate how recombinant MASP-1 (rMASP-1) can modify the pattern of P-selectin, E-selectin, ICAM-1, ICAM-2, and VCAM-1 adhesion molecules in human umbilical vein endothelial cells (HUVEC), and whether these changes can enhance the adherence between endothelial cells and neutrophil granulocyte model cells (differentiated PLB-985). We found that HUVECs activated by rMASP-1 decreased the expression of ICAM-2 and increased that of E-selectin, whereas ICAM-1, VCAM-1 and P-selectin expression remained unchanged. Furthermore, these changes resulted in increased adherence between differentiated PLB-985 cells and endothelial cells. Our finding suggests that complement MASP-1 can increase adhesion between neutrophils and endothelial cells in a direct fashion. This is in agreement with our previous finding that MASP-1 increases the production of pro-inflammatory cytokines (such as IL-6 and IL-8) and chemotaxis, and may thereby boost neutrophil functions. This newly described cooperation between complement lectin pathway and neutrophils via endothelial cells may be an effective tool to enhance the antimicrobial immune response.

Early High-dosage Atorvastatin Treatment Improved Serum Immune-inflammatory Markers and Functional Outcome in Acute Ischemic Strokes Classified as Large Artery Atherosclerotic Stroke: A Randomized Trial.

Statins have beneficial effects on cerebral circulation and brain parenchyma during ischemic stroke and reperfusion. The primary hypothesis of this randomized parallel trial was that treatment with 80 mg/day of atorvastatin administered early at admission after acute atherosclerotic ischemic stroke could reduce serum levels of markers of immune-inflammatory activation of the acute phase and that this immune-inflammatory modulation could have a possible effect on prognosis of ischemic stroke evaluated by some outcome indicators. We enrolled 42 patients with acute ischemic stroke classified as large arteries atherosclerosis stroke (LAAS) randomly assigned in a randomized parallel trial to the following groups: Group A, 22 patients treated with atorvastatin 80 mg (once-daily) from admission day until discharge; Group B, 20 patients not treated with atorvastatin 80 mg until discharge, and after discharge, treatment with atorvastatin has been started. At 72 hours and at 7 days after acute ischemic stroke, subjects of group A showed significantly lower plasma levels of tumor necrosis factor-α, interleukin (IL)-6, vascular cell adhesion molecule-1, whereas no significant difference with regard to plasma levels of IL-10, E-Selectin, and P-Selectin was observed between the 2 groups. At 72 hours and 7 days after admission, stroke patients treated with atorvastatin 80 mg in comparison with stroke subjects not treated with atorvastatin showed a significantly lower mean National Institutes of Health Stroke Scale and modified Rankin scores. Our findings provide the first evidence that atorvastatin acutely administered immediately after an atherosclerotic ischemic stroke exerts a lowering effect on immune-inflammatory activation of the acute phase of stroke and that its early use is associated to a better functional and prognostic profile.