Activation of vascular endothelial cells by IL-1alpha released by epithelial cells infected with respiratory syncytial virus

Although pulmonary inflammation is a serious, sometimes life-threatening, consequence of respiratory syncytial virus (RSV) infection, the mechanisms involved are not well understood. Since the process of inflammation is initiated by a complex series of events including the activation of specific adhesion molecules on vascular endothelium, we searched for endothelial cell-activating factors released from RSV-infected epithelial cells. We demonstrate here that vascular endothelial cells exposed to culture supernatants from RSV-infected pulmonary epithelial A549 cells are activated to express increased cell surface ICAM-1, and to a lesser extent, VCAM-1 and E-selectin. IL-1alpha was identified as the predominant endothelial cell-activating factor by pretreating epithelial cell supernatants with anti-IL-1alpha antibody. The preferential upregulation of endothelial ICAM-1 (relative to VCAM-1 and E-selectin) by RSV-infected epithelial cell supernatants was replicated by recombinant IL-1alpha thus confirming IL-1alpha as a major endothelial cell-activating cytokine released by RSV-infected epithelial cells. Il-1alpha mediated endothelial cell activation is thus a likely contributory event in the initiation of leukocyte inflammation associated with RSV infection.     

The influence of garlic (Allium sativum) extract on interleukin 1alpha-induced expression of endothelial intercellular adhesion molecule-1 and vascular cell adhesion molecule-1.

Inflammation plays an important role in both the initiation of atherosclerosis and development of atherothrombotic events. The adherence of leukocytes/monocytes to the endothelium is an early event in atherogenesis. Phytotherapeutica as garlic and garlic extracts were shown to have beneficial modulating effects in patients with atherosclerotic disease. The aim of this study was to evaluate in vitro the influence of water-soluble garlic (Allium sativum) extract on the cytokine-induced expression of endothelial leukocyte adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1, CD54) and vascular cell adhesion molecule-1 (VCAM-1, CD106). Cytokine-induced expression of cellular adhesion molecules was measured on primary human coronary artery endothelial cell (HCAEC) cultures. HCAEC were cultured in microvascular endothelial cell growth medium and preincubated with garlic extract at various concentrations (0.25-4.0 mg/ml), after which human interleukin-1alpha (IL-1alpha, 10 ng/ml) was added for 1 day. Fluorescein isothiocyanate (FITC)-labeled anti-ICAM-1 and FITC-labeled anti-VCAM-1 were used to analyze the IL-1alpha-induced expression of ICAM-1 and VCAM-1 by flow cytometry. Incubation of HCAEC with garlic extract significantly decreased ICAM-1 and VCAM-1 expression induced by IL-1alpha. In addition, we examined the effects of garlic extract on the adhesion of monocytes to endothelial cells, using the monocytic U937 cell line. The presence of garlic extract significantly inhibited the adhesion of monocytes to IL-1alpha-stimulated endothelial cells. These results indicate that garlic extract modulates the expression of ICAM-1 and VCAM-1, thus potentially contributing to the beneficial effects traditionally attributed to garlic.     

Regulation of vascular cell adhesion molecule 1 on human dermal microvascular endothelial cells.

Vascular endothelial cell adhesion molecule 1 (VCAM-1) is an adherence molecule that is induced on endothelial cells by cytokine stimulation and can mediate binding of lymphocytes or tumor cells to endothelium. Because these interactions often occur at the level of the microvasculature, we have examined the regulation of expression of VCAM-1 in human dermal microvascular endothelial cells (HDMEC) and compared it to the regulation of VCAM-1 in large vessel human umbilical vein endothelial cells (HUVEC). Both cell populations were judged pure as assessed by expression of von Willebrand factor and uptake of acetylated low density lipoprotein. Expression of VCAM-1 was not detectable on either unstimulated HDMEC or HUVEC when assessed by ELISA or flow cytometry. Stimulation of either HDMEC or HUVEC with TNF-alpha resulted in a time- and dose-dependent induction of VCAM-1. However, although TNF-alpha-induced cell surface and mRNA expression of VCAM-1 in HDMEC was transient, peaking after 16 h of stimulation, TNF stimulation led to persistently elevated cell surface expression of VCAM-1 on HUVEC. IL-1 alpha also induced cell surface expression of VCAM-1 on HUVEC in a time- and dose-dependent manner, but stimulation of HDMEC with IL-1 alpha at doses up to 1000 U/ml failed to induce significant cell surface expression. However, IL-1 alpha induced time- and dose-dependent increases in ICAM-1 on HDMEC. Similarly, IL-4 induced VCAM-1 expression and augmented TNF-alpha-induced expression on HUVEC but did not affect VCAM-1 expression on HDMEC. Binding of Ramos cells to cytokine-stimulated endothelial cell monolayers correlated with VCAM-1 induction. Increased binding was seen after stimulation of HDMEC with TNF-alpha, which was blocked by anti-VCAM-1 mAb, but no increases in binding were noted after stimulation of HDMEC monolayers with IL-1 alpha. These data provide additional evidence for the existence of endothelial cell heterogeneity and differences in cell adhesion molecule regulation on endothelial cells derived from different vascular beds.     

A novel role for interleukin-18 in adhesion molecule induction through NF kappa B and phosphatidylinositol (PI) 3-kinase-dependent signal transduction pathways

Interleukin-18 (IL-18) is a novel proinflammatory cytokine found in serum and joints of patients with rheumatoid arthritis (RA). We studied a novel role for IL-18 in mediating cell adhesion, a vital component of the inflammation found in RA and other inflammatory diseases. We examined the expression of cellular cell adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) on endothelial cells and RA synovial fibroblasts using flow cytometry. Adhesion of the monocyte-like cell line HL-60 to endothelial cells was determined by immunofluorescence. IL-18 significantly enhanced ICAM-1 and VCAM-1 expression on endothelial cells and RA synovial fibroblasts. In addition, IL-18 induced E-selectin expression on endothelial cells and promoted the adhesion of HL-60 cells to IL-18-stimulated endothelial cells. Neutralizing anti-VCAM-1 and anti-E-selectin could completely inhibit HL-60 adherence to endothelial cells. IL-18-induced adhesion molecule expression appears to be mediated through nuclear factor kappa B (NF kappa B) and phosphatidyl-inositol 3 kinase (PI 3-kinase) since addition of inhibitors to either NF kappa B (pyrrolidine dithiocarbamate and N-acetyl-l-cysteine) or PI 3-kinase (LY294002) inhibited RA synovial fibroblast VCAM-1 expression by 50 to 60%. Addition of both inhibitors resulted in inhibition of VCAM-1 expression by 85%. In conclusion, the ability of IL-18 to induce adhesion molecule expression on endothelial cells and RA synovial fibroblasts indicates that IL-18 may contribute to RA joint inflammation by enhancing the recruitment of leukocytes into the joint. IL-18 requires NF kappa B as well as PI 3-kinase to induce VCAM-1 on RA synovial fibroblasts, suggesting that there may be two distinct pathways in IL-18-induced adhesion molecule expression.     

1,4-dihydroxyxanthone modulates the adhesive property of endothelial cells by inhibiting intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin

Cell adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM-1) and E-selectin, play important roles in the recruitment of leukocytes to the site of inflammation. Blocking the expression of these molecules or preventing their interaction with the receptors has been shown to be important in controlling various inflammatory diseases. These cell adhesion molecules are induced on endothelial cells by various proinflammatory cytokines like IL-1beta and TNF-alpha and also by bacterial LPS. We demonstrate here that 1,4-Dihydroxyxanthone (1,4 DHX) inhibits the expression of cell adhesion molecules, such as ICAM-1, VCAM-1 and E-selectin, on endothelial cells in a concentration and time dependent manner. The inhibition by 1,4 DHX is reversible. On further analysis, our results also show that 1,4 DHX inhibits the adhesion of peripheral neutrophils to the endothelial cell monolayers. 1,4 DHX, therefore, could be used as a novel target for controlling various pathological conditions associated with upregulation of endothelial leukocyte adhesion molecules.     

Mast cells are potent regulators of endothelial cell adhesion molecule ICAM-1 and VCAM-1 expression

To investigate the possible role of mast cells (MC) in regulating leukocyte adhesion to vascular endothelial cells (EC), microvascular and macrovascular EC were exposed to activated MC or MC conditioned medium (MCCM). Expression of intercellular and vascular adhesion molecules (ICAM-1 and VCAM-1) on EC was monitored. Incubation of human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells (HUVEC) with activated MC or MCCM markedly increased ICAM-1 and VCAM-1 surface expression, noted as éarly as 4 hr. Maximal levels were observed at 16 hr followed by a general decline over 48 hr. A dose-dependent response was noted using incremental dilutions of MCCM or by varying the number of MC in coculture with EC. At a ratio as low as 1:1,000 of MC:EC, increased ICAM-1 was observed. The ICAM-1 upregulation by MCCM was >90% neutralized by antibody to tumor necrosis factor alpha (TNF-α), suggesting that MC release of this cytokine contributes significantly to inducing EC adhesiveness. VCAM-1 expression enhanced by MCCM was partly neutralized (70%) by antibody to TNF-α; thus other substances released by MC may contribute to VCAM-1 expression. Northern blot analysis demonstrated MCCM upregulated ICAM-1 and VCAM-1 mRNA in both HDMEC and HUVEC. To evaluate the function of MCCM-enhanced EC adhesion molecules, T cells isolated from normal human donors were used in a cell adhesion assay. T-cell binding to EC was increased significantly after exposure of EC to MCCM, and inhibited by antibodies to ICAM-1 or VCAM-1. Intradermal injection of allergen in human atopic volunteers known to develop late-phase allergic reactions led to marked expression of both ICAM-1 and VCAM-1 at 6 hr, as demonstrated by immunohistochemistry. These studies indicate that MC play a critical role in regulating the expression of EC adhesion molecules, ICAM-1 and VCAM-1, and thus augment inflammatory responses by upregulating leukocyte binding. © 1995 Wiley-Liss Inc.     

Proinflammatory and Th2-derived cytokines modulate CD40-mediated expression of inflammatory mediators in airway epithelia: implications for the role of epithelial CD40 in airway inflammation

Cytokines produced by activated macrophages and Th2 cells within the lung play a key role in asthma-associated airway inflammation. Additionally, recent studies suggest that the molecule CD40 modulates lung immune responses. Because airway epithelial cells can act as immune effector cells through the expression of inflammatory mediators, the epithelium is now considered important in the generation of asthma-associated inflammation. Therefore, the goal of the present study was to examine the effects of proinflammatory and Th2-derived cytokines on the function of CD40 in airway epithelia. The results show that airway epithelial cells express CD40 and that engagement of epithelial CD40 induces a significant increase in expression of the chemokines RANTES, monocyte chemoattractant protein (MCP-1), and IL-8 and the adhesion molecule ICAM-1. Cross-linking epithelial CD40 had no effect on expression of the adhesion molecule VCAM-1. The proinflammatory cytokines TNF-alpha and IL-1beta and the Th2-derived cytokines IL-4 and IL-13 modulated the positive effects of CD40 engagement on inflammatory mediator expression in airway epithelial cells. Importantly, CD40 ligation enhanced the sensitivity of airway epithelial cells to the effects of TNF-alpha and/or IL-1beta on expression of RANTES, MCP-1, IL-8, and VCAM-1. In contrast, neither IL-4 nor IL-13 modified the effects of CD40 engagement on the expression of RANTES, MCP-1, IL-8, or VCAM-1; however, both IL-4 and IL-13 attenuated the effects of CD40 cross-linking on ICAM-1 expression. Together, these findings suggest that interactions between CD40-responsive airway epithelial cells and CD40 ligand+ leukocytes, such as activated T cells, eosinophils, and mast cells, modulate asthma-associated airway inflammation.     

IKKalpha and IKKbeta function in TNFalpha-stimulated adhesion molecule expression in human aortic smooth muscle cells

The role of NFkappaB and it's upstream kinases in regulating adhesion molecule expression in the smooth muscle of the vasculature remains controversial. We therefore examined the effect of blocking the NFkappaB pathway on TNFalpha-stimulated ICAM-1 and VCAM-1 expression in primary cultures of human aortic smooth muscle cells using an adenoviral wild-type IkappaB alpha construct (Ad.IkappaB alpha) and dominant-negative IKKalpha (Ad.IKKalpha+/-) and IKKbeta (Ad.IKKbeta+/-) constructs. Ad.IkappaB alpha treatment was found to block NFkappaB DNA-binding, and thereby completely prevent TNFalpha-stimulated ICAM-1 and VCAM-1 expression without influencing IKK activity. Ad.IKKbeta+/- treatment completely inhibited TNFalpha-stimulated IKK kinase activity, IkappaB alpha degradation and NFkappaB DNA-binding in addition to completely blocking TNFalpha-stimulated ICAM-1 and VCAM-1 expression. Ad.IKKalpha+/- treatment however had no detectable effect on NFkappaB DNA-binding or ICAM-1 and VCAM-1 expression. Our results demonstrate that TNFalpha-stimulated ICAM-1 and VCAM-1 expression in human aortic smooth muscle cells is NFkappaB-dependent, that IKKbeta is a suitable target for drug therapy and Ad.IKKbeta+/- an effective inhibitor of TNFalpha-stimulated ICAM-1 and VCAM-1 expression.     

Effects of tumor necrosis factor, lipopolysaccharide, and IL-4 on the expression of vascular cell adhesion molecule-1 in vivo. Correlation with CD3+ T cell infiltration.

We have injected human TNF, LPS, and IL-4 into the skin of baboons to examine regulation of endothelial leukocyte adhesion molecules (ELAM) in vivo and to determine which endothelial adhesion molecules correlate temporally and spatially with cytokine-induced T cell infiltration. The expression of adhesion molecules ELAM-1 (E-selectin), VCAM-1, and ICAM-1 (CD54) were quantified by immunocytochemical staining of frozen sections obtained from skin biopsies; T cell infiltration was measured by immunocytochemical staining of CD3+ T cells in serial sections. We found that injection of TNF causes late (24 to 48 h) T cell infiltration whereas injection of LPS, in doses that do not cause tissue necrosis, does not. The ability of TNF (but not LPS) to recruit T cells correlates with the ability of TNF to cause sustained endothelial cell adhesion molecule expression. Expression of VCAM-1 on post-capillary venules showed the highest degree of spatial localization with infiltrates. IL-4, although not proinflammatory by itself, can cause T cell infiltration in combination with an ineffective dose of TNF. The ability of IL-4 to augment TNF-induced inflammation best correlates with the ability of the combination of IL-4 and TNF to increase endothelial VCAM-1 expression. In contrast, IL-4 does not promote T cell infiltration or endothelial VCAM-1 expression in combination with LPS. In cytokine-injected tissues, VCAM-1 is also expressed on connective tissue cells other than endothelium, including smooth muscle and perineural cells, where it is induced by cytokines in parallel with endothelial VCAM-1. Overall, our data support the hypothesis that endothelial VCAM-1 expression contributes to T cell extravasation at sites of inflammation. Furthermore, we find that IL-4, a product a Ag-activated T cells, can interact with TNF to selectively promote VCAM-1 expression and the development of T cell-rich infiltrates, characteristic of Ag-induced inflammatory reactions.     

Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin through nuclear factor-kappa B activation in endothelial cells

Vascular endothelial growth factor (VEGF) induces adhesion molecules on endothelial cells during inflammation. Here we examined the mechanisms underlying VEGF-stimulated expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin in human umbilical vein endothelial cells. VEGF (20 ng/ml) increased expression of ICAM-1, VCAM-1, and E-selectin mRNAs in a time-dependent manner. These effects were significantly suppressed by Flk-1/kinase-insert domain containing receptor (KDR) antagonist and by inhibitors of phospholipase C, nuclear factor (NF)-kappaB, sphingosine kinase, and protein kinase C, but they were not affected by inhibitors of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1/2 or nitric-oxide synthase. Unexpectedly, the phosphatidylinositol (PI) 3'-kinase inhibitor wortmannin enhanced both basal and VEGF-stimulated adhesion molecule expression, whereas insulin, a PI 3'-kinase activator, suppressed both basal and VEGF-stimulated expression. Gel shift analysis revealed that VEGF stimulated NF-kappaB activity. This effect was inhibited by phospholipase C, NF-kappaB, or protein kinase C inhibitor. VEGF increased VCAM-1 and ICAM-1 protein levels and increased leukocyte adhesiveness in a NF-kappaB-dependent manner. These results suggest that VEGF-stimulated expression of ICAM-1, VCAM-1, and E-selectin mRNAs was mainly through NF-kappaB activation with PI 3'-kinase-mediated suppression, but was independent of nitric oxide and MEK. Thus, VEGF simultaneously activates two signal transduction pathways that have opposite functions in the induction of adhesion molecule expression. The existence of parallel inverse signaling implies that the induction of adhesion molecule expression by VEGF is very finely regulated.     

ESM-1 promotes adhesion between monocytes and endothelial cells under intermittent hypoxia

Intermittent hypoxia (IH), the key property of obstructive sleep apnea (OSA), is closely associated with endothelial dysfunction. Endothelial-cell-specific molecule-1 (ESM-1, Endocan) is a novel, reported molecule linked to endothelial dysfunction. The aim of this study is to evaluate the effect of IH on ESM-1 expression and the role of ESM-1 in endothelial dysfunction. We found that serum concentration of ESM-1, inter-cellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) is significantly higher in patients with OSA than healthy volunteers (p < 0.01). The expression of ESM-1, hypoxia-inducible factor-1 alpha (HIF-1α), and vascular endothelial growth factor (VEGF) was significantly increased in human umbilical vein endothelial cells (HUVECs) by treated IH in a time-dependent manner. HIF-1α short hairpin RNA and vascular endothelial growth factor receptor (VEGFR) inhibitor inhibited the expression of ESM-1 in HUVECs. ICAM-1 and VCAM-1 expressions were significantly enhanced under IH status, accompanied by increased monocyte–endothelial cell adhesion rate ( p < 0.001). Accordingly, ESM-1 silencing decreased the expression of ICAM-1 and VCAM-1 in HUVECs, whereas ESM-1 treatment significantly enhanced ICAM-1 expression accompanied by increasing adhesion ability. ESM-1 is significantly upregulated by the HIF-1α/VEGF pathway under IH in endothelial cells, playing a critical role in enhancing adhesion between monocytes and endothelial cells, which might be a potential target for IH-induced endothelial dysfunction.     

Trophoblast interactions with endothelial cells are increased by interleukin-1beta and tumour necrosis factor alpha and involve vascular cell adhesion molecule-1 and alpha4beta1

Interactions between fetal extravillous trophoblast cells and maternal uterine cells are of critical importance in successful placentation. In the first trimester, trophoblasts invade the uterine environment and reach the spiral arteries where they interact with vascular cells; however, little is known of the nature of these interactions. We have developed a fluorescent binding assay to investigate the contact between trophoblasts and endothelial cells and to determine its regulation by cytokines and adhesion molecules. Stimulation of an endothelial cell line (SGHEC-7) with interleukin-1beta or tumour necrosis factor-alpha significantly increased adhesion of the first-trimester extravillous trophoblast-derived cell line, SGHPL-4. Using blocking antibodies, vascular cell adhesion molecule-1 (VCAM-1) and integrin alpha4beta1 (VLA-4), but not intercellular adhesion molecule-1 (ICAM-1), were shown to be important in trophoblast binding to activated endothelial cells. SGHPL-4 cells were shown to express HLA-G, alpha4beta1 and ICAM-1 at high levels and LFA-1 and VCAM-1 at lower levels. ICAM-1 and VCAM-1 are expressed on SGHEC-7 cells and their expression was confirmed on primary decidual endothelial cells. In conclusion, we have demonstrated the importance of VCAM-1 and alpha4beta1 in trophoblasts-endothelial interactions. Improved knowledge of the nature of these fetal-maternal interactions will have implications for understanding situations when placentation is compromised.     

Butyrate inhibits cytokine-induced VCAM-1 and ICAM-1 expression in cultured endothelial cells: the role of NF-kappaB and PPARalpha

Adhesion and migration of leukocytes into the surrounding tissues is a crucial step in inflammation, immunity, and atherogenesis. Expression of cell adhesion molecules by endothelial cells plays a leading role in this process. Butyrate, a natural short-chain fatty acid produced by bacterial fermentation of dietary fiber, has been attributed with anti-inflammatory activity in inflammatory bowel disease. Butyrate in vitro is active in colonocytes and several other cell types. We have studied the effect of butyrate on expression of endothelial leukocyte adhesion molecules by cytokine-stimulated human umbilical vein endothelial cells (HUVEC). Pretreatment of HUVEC with butyrate-inhibited tumor necrosis factor-alpha (TNFalpha)-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) in a time and concentration-dependent manner. Butyrate at 10 mM/L inhibited interleukin-1 (IL-1)-stimulated VCAM-1 and ICAM-1 expression. The effect of butyrate on cytokine-stimulated VCAM-1 expression was more pronounced than in the case of ICAM-1. Butyrate decreased TNFalpha-induced expression of mRNA for VCAM-1 and ICAM-1. Suppressed expression of VCAM-1 and ICAM-1 was associated with reduced adherence of monocytes and lymphocytes to cytokine-stimulated HUVEC. Butyrate inhibited TNFalpha-induced activation of nuclear factor-kappaB (NF-kappaB) in HUVEC. Finally, butyrate enhanced peroxisome proliferator-activated receptor-alpha (PPARalpha) expression in HUVEC. These results demonstrate that butyrate may have anti-inflammatory properties not only in colonocytes but also in endothelial cells. The anti-inflammatory and (perhaps) antiatherogenic properties of butyrate may partly be attributed to an effect on activation of NF-kappaB and PPARalpha and to the associated expression of VCAM-1 and ICAM-1. The present findings support further investigations on the therapeutic benefits of butyrate in several pathological events involving leukocyte recruitment.     

Expression of cell adhesion molecules and lymphocyte-endothelium interaction under simulated hypogravity in vitro

Using histochemical staining and FACS-analysis we have studied the basal and TNF-alpha induced expression of E-selectin, ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (ECs) exposed to simulated hypogravity. Control ECs did not contain detectable amounts of E-selectin or VCAM-1 but were ICAM-1 positive. As soon as after 6-8 hrs of clinorotation at 5 RPM the cellular content of ICAM- 1 increased. Moreover, hypogravity potentiated the effect of inflammatory cytokines (TNF-alpha and IL-1) on ICAM-1 expression. No increase in E-selectin or VCAM-1 expression was observed in ECs exposed to hypogravity itself. However, hypogravity reduced E-selectin and VCAM-1 expression in cell cultures activated by cytokines, more visible at their low (5-10 U/ml) concentrations. Both, control and clinorotated ECs poorly supported spontaneous lymphocyte adhesion; the adhesion of PMA-activated leukocytes was 15-20-fold higher. The interaction of unstimulated lymphocytes with cytokine-activated endothelium was more noticeable but significantly lower in cultures exposed to hypogravity. Activated blood cells interacted with endothelium more effectively, particularly, under hypogravity. Obtained results suggest that EC adhesion molecule expression and endothelium-lymphocyte interaction are altered under simulated hypogravity conditions in direction of increase of endotlielial adhesiveness for activated blood cells.     

Induction of cell adhesion molecules, E-selectin, VCAM-1 and ICAM-1, in a co-culture of human endothelial and smooth muscle cells

Using immunofluorescence and flow cytometry, we studied the surface expression of cell adhesion molecules, E-selectin, VCAM-1 and ICAM-1, in human umbilical vein endothelial cells (HUVEC) co-cultured with human aortic intimal smooth muscle cells (SMC). It was found that inactivated HUVEC constitutively expressed only ICAM-1. After 3-4 h of co-culturing with SMC in the Transwell system we observed the appearance of E-selectin and VCAM-1, and the increase of ICAM-1 content on the cell surface. In all the cases, the maximum expression of these molecules (85-100% of positively stained cells) was detected within 18-24 h after co-culturing. Similar effect was exerted by SMC-conditioned culture medium, whose action well compared with that of a direct addition of interleukin-1 to EC at a concentration of 5-10 u/ml. The obtained data suggest that the cytokines secreted by SMC may participate in the regulation of endothelial cell adhesion molecule expression, and influence cell accumulation in sites of inflammation, immune disorders, etc.     

Relaxin inhibits early steps in vascular inflammation

Increased expression of endothelial adhesion molecules, high levels of the monocyte chemoattractant protein-1 (MCP-1) and enhanced VLA4 integrin/VCAM-1 and CCR-2/MCP-1 interactions are initial steps in vascular inflammation. We sought to determine whether relaxin, a potent vasodilatory and anti-fibrotic agent, mitigates these early events compromising endothelial integrity. The effect of relaxin coincubation on the TNF-α-stimulated expression of the adhesion molecules VCAM-1, ICAM-1 and E-selectin; the MCP-1 expression by human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (HAoSMC); as well as on direct monocyte–endothelium cell adhesion was quantified by ELISA or adhesion assay. CCR-2 and PECAM expression on HUVEC and THP-1 monocytes was investigated by FACS analysis. Relaxin treatment suppressed significantly TNF-α-induced upregulation of VCAM-1 and PECAM, CCR-2, and MCP-1 levels and direct monocyte adhesion to HUVEC. Our findings identify relaxin as a promising inhibitory factor in early vascular inflammation. By attenuating the upregulation of VCAM-1, key adhesion molecule in early vascular inflammation, and of MCP-1, a chemokine pivotal to monocyte recruitment, relaxin decreased initial monocyte–endothelium contact. This may be of relevance for the prevention and treatment of atherosclerosis and of other pro-inflammatory states.     

Preconditioning with endoplasmic reticulum stress mitigates retinal endothelial inflammation via activation of X-box binding protein 1

Endoplasmic reticulum (ER) stress is widely implicated in various pathological conditions such as diabetes. Previously, we reported that enhanced ER stress contributes to inflammation and vascular damage in diabetic and ischemia-induced retinopathy. However, the exact role of the signaling pathways activated by ER stress in vascular inflammation remains poorly understood. In the present study, we investigated the role of X-box binding protein 1 (XBP1) in retinal adhesion molecule expression, leukostasis, and vascular leakage. Exposure of human retinal endothelial cells to low dose ER stress inducers resulted in a robust activation of XBP1 but did not affect inflammatory gene expression. However, ER stress preconditioning almost completely abolished TNF-α-elicited NF-κB activation and adhesion molecule ICAM-1 and VCAM-1 expression. Pharmaceutical inhibition of XBP1 activation or knockdown of XBP1 by siRNA markedly attenuated the effects of preconditioning on inflammation. Moreover, loss of XBP1 led to an increase in ICAM-1 and VCAM-1 expression. Conversely, overexpression of spliced XBP1 attenuated TNF-α-induced phosphorylation of IKK, IκBα, and NF-κB p65, accompanied by decreased NF-κB activity and reduced adhesion molecule expression. Finally, in vivo studies show that activation of XBP1 by ER stress preconditioning prevents TNF-α-induced ICAM-1 and VCAM-1 expression, leukostasis, and vascular leakage in mouse retinas. These results collectively indicate a protective effect of ER stress preconditioning against retinal endothelial inflammation, which is likely through activation of XBP1-mediated unfolded protein response (UPR) and inhibition of NF-κB activation.     

Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

Serum retinol-binding protein 4 (RBP4) is the sole specific vitamin A (retinol) transporter in blood. Elevation of serum RBP4 in patients has been linked to cardiovascular disease and diabetic retinopathy. However, the significance of RBP4 elevation in the pathogenesis of these vascular diseases is unknown. Here we show that RBP4 induces inflammation in primary human retinal capillary endothelial cells (HRCEC) and human umbilical vein endothelial cells (HUVEC) by stimulating expression of proinflammatory molecules involved in leukocyte recruitment and adherence to endothelium, including vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-selectin, monocyte chemoattractant protein 1 (MCP-1), and interleukin-6 (IL-6). We demonstrate that these novel effects of RBP4 are independent of retinol and the RBP4 membrane receptor STRA6 and occur in part via activation of NADPH oxidase and NF-κB. Importantly, retinol-free RBP4 (apo-RBP4) was as potent as retinol-bound RBP4 (holo-RBP4) in inducing proinflammatory molecules in both HRCEC and HUVEC. These studies reveal that RBP4 elevation can directly contribute to endothelial inflammation and therefore may play a causative role in the development or progression of vascular inflammation during cardiovascular disease and microvascular complications of diabetes.     

The integrin alpha9beta1 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1

The integrin alpha9beta1 has been shown to be widely expressed on smooth muscle and epithelial cells, and to mediate adhesion to the extracellular matrix proteins osteopontin and tenascin-C. We have found that the peptide sequence this integrin recognizes in tenascin-C is highly homologous to the sequence recognized by the closely related integrin alpha4beta1, in the inducible endothelial ligand, vascular cell adhesion mole-cule-1 (VCAM-1). We therefore sought to determine whether alpha9beta1 also recognizes VCAM-1, and whether any such interaction would be biologically significant. In this report, we demonstrate that alpha9beta1 mediates stable cell adhesion to recombinant VCAM-1 and to VCAM-1 induced on human umbilical vein endothelial cells by tumor necrosis factor-alpha. Furthermore, we show that alpha9beta1 is highly and selectively expressed on neutrophils and is critical for neutrophil migration on VCAM-1 and tenascin-C. Finally, alpha9beta1 and alpha4 integrins contribute to neutrophil chemotaxis across activated endothelial monolayers. These observations suggest a possible role for alpha9beta1/VCAM-1 interactions in extravasation of neutrophils at sites of acute inflammation.