Pharmacological targeting of HSP90 with 17-AAG induces apoptosis of myogenic cells through activation of the intrinsic pathway

Endothelial inflammation and monocyte plays an essential role in the initiation and progression of atherosclerosis. Ghrelin is beneficial for atherosclerosis progression. However, the detailed and precise molecular mechanisms of how ghrelin regulates endothelial inflammation are not clear. In this study, we investigated the regulation mechanism of ghrelin on TNF-α-activated endothelial inflammation and monocyte adhesion. It was found that TNF-α-induced monocyte adhesion on HUVEC was significantly attenuated by ghrelin. Furthermore, we found that ghrelin effectively suppressed TNF-α-induced inflammatory factors’ (including ICAM-1, VCAM-1, MCP-1, and IL-1β) expression through inhibiting AMPK phosphorylation and p65 expression both in HUVEC and THP-1. This phenomenon was further demonstrated by using AMPK agonist AICAR and inhibitor compound C, respectively. Our findings suggest that ghrelin may mediate TNF-α-induced endothelial inflammation and monocyte adhesion, in part via AMPK/NF-κB signaling pathway. These novel anti-inflammatory and immunoregulatory actions of ghrelin may play a certain role in understanding the formation and development of atherosclerosis.     

Ginsenoside metabolite compound K differentially antagonizing tumor necrosis factor-α-induced monocyte-endothelial trafficking

Human leukocyte endothelial adhesion and transmigration occur in the early stage of the pathogenesis of atherosclerosis. Vascular endothelial cells are targeted by pro-inflammatory cytokines modulating many gene proteins responsible for cell adhesion, thrombosis and inflammatory responses. This study examined the potential of compound K to inhibit the pro-inflammatory cytokine TNF-α induction of monocyte adhesion onto TNF-α-activated human umbilical vein endothelial cells (HUVEC). HUVEC were cultured with 10 ng/ml TNF-α with individual ginsenosides of Rb1, Rc, Re, Rh1 and compound K (CK). Ginsenosides at doses of ?50 μM did not show any cytotoxicity. TNF-α induced THP-1 monocyte adhesion to HUVEC, and such induction was attenuated by Rh1 and CK. Consistently, CK suppressed TNF-α-induced expression of HUVEC adhesion molecules of VCAM-1, ICAM-1 and E-selectin, and also Rh1 showed a substantial inhibition. Rh1 and CK dampened induction of counter-receptors, α4/β1 integrin VLA-4 and αL/β2 integrin LFA-1 in TNF-α-treated THP-1 cells. Additionally, CK diminished THP-1 secretion of MMP-9 required during transmigration, inhibiting transendothelial migration of THP-1 cells. CK blunted TNF-α-promoted IL-8 secretion of HUVEC and CXCR1 expression of THP-1 monocytes. Furthermore, TNF-α-activated endothelial IκB phosphorylation and NF-κB nuclear translocation were disturbed by CK, and TNF-α induction of α4/β1 integrin was abrogated by the NF-κB inhibitor SN50. These results demonstrate that CK exerts anti-atherogenic activity with blocking leukocyte endothelial interaction and transmigration through negatively mediating NF-κB signaling.     

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.     

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.     

Salusin-β accelerates inflammatory responses in vascular endothelial cells via NF-κB signaling in LDL receptor-deficient mice in vivo and HUVECs in vitro

The bioactive peptide salusin-β is highly expressed in human atheromas; additionally, infusion of antiserum against salusin-β suppresses the development of atherosclerosis in atherogenic mice. This study examined the roles of salusin-β in vascular inflammation during atherogenesis. Infusion of antiserum against salusin-β attenuated the induction of VCAM-1, monocyte chemoattractant protein (MCP)-1, and IL-1β and as well as nuclear translocation of NF-κB in aortic endothelial cells (ECs) of LDL receptor-deficient mice, which led to the prevention of monocyte adhesion to aortic ECs. In vitro experiments indicated that salusin-β directly enhances the expression levels of proinflammatory molecules, including VCAM-1, MCP-1, IL-1β, and NADPH oxidase 2, as well as THP-1 monocyte adhesion to cultured human umbilical vein ECs (HUVECs). Both salusin-β-induced VCAM-1 induction and monocyte/HUVEC adhesion were suppressed by pharmacological inhibitors of NF-κB, e.g., Bay 11-7682 and curcumin. Furthermore, the VCAM-1 induction was significantly prevented by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002, whereas it was accelerated by the ERK inhibitor, U-0126. Treatment of HUVECs with salusin-β, but not with salusin-α, accelerated oxidative stress and nuclear translocation of NF-κB as well as phosphorylation and degradation of IκB-α, an endogenous inhibitor of NF-κB. Thus, salusin-β enhanced monocyte adhesion to vascular ECs through NF-κB-mediated inflammatory responses in ECs, which can be modified by PI3K or ERK signals. These findings are suggestive of a novel role of salusin-β in atherogenesis.     

Lipoxin A4 inhibits immune cell binding to salivary epithelium and vascular endothelium

Lipoxins are formed by leukocytes during cell-cell interactions with epithelial or endothelial cells. Native lipoxin A(4) (LXA(4)) binds to the G protein-coupled lipoxin receptors formyl peptide receptor 2 (FPR2)/ALX and CysLT1. Furthermore, LXA(4) inhibits recruitment of neutrophils, by attenuating chemotaxis, adhesion, and transmigration across vascular endothelial cells. LXA(4) thus appears to serve as an endogenous "stop signal" for immune cell-mediated tissue injury (Serhan CN; Annu Rev Immunol 25: 101-137, 2007). The role of LXA(4) has not been addressed in salivary epithelium, and little is known about its effects on vascular endothelium. Here, we determined that interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) receptor activation in vascular endothelium and salivary epithelium upregulated the expression of adhesion molecules that facilitates the binding of immune cells. We hypothesize that the activation of the ALX/FPR2 and/or CysLT1 receptors by LXA(4) decreases this cytokine-mediated upregulation of cell adhesion molecules that enhance lymphocyte binding to both the vascular endothelium and salivary epithelium. In agreement with this hypothesis, we observed that nanomolar concentrations of LXA(4) blocked IL-1β- and TNF-α-mediated upregulation of E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVECs). Binding of Jurkat cells to stimulated HUVECs was abrogated by LXA(4). Furthermore, LXA(4) preincubation with human submandibular gland cell line (HSG) also blocked TNF-α-mediated upregulation of vascular cell adhesion molecule-1 (VCAM-1) in these cells, and it reduced lymphocyte adhesion. These findings suggest that ALX/FPR2 and/or CysLT1 receptor activation in endothelial and epithelial cells blocks cytokine-induced adhesion molecule expression and consequent binding of lymphocytes, a critical event in the pathogenesis of Sj?gren's syndrome (SS).     

Expression of Cell Adhesion Molecules, their Ligands and Tumour Necrosis Factor α in the Liver of Patients with Metastatic Gastrointestinal Carcinomas

The expression of the following cell adhesion molecules, their β1 and β2 integrin ligands and the cytokine tumour necrosis factor-α (TNF-α) was investigated by light and electron microscope immunohistochemistry in the liver tissue in 20 patients with colorectal and gastric cancer also presenting with liver metastases: intercellular adhesion molecule-1 (ICAM-1), vascular endothelial adhesion molecule-1 (VCAM-1), E-selectin, leucocyte function-associated antigen-1 (LFA-1), macrophage antigen-1 (Mac-1), and very late antigen-4 (VLA-4). We have found a parallel enhancement of the adhesion molecules and of TNF-α in liver sinusoids surrounding metastases. The expression of ICAM-1 was enhanced on sinusoidal cells in all zones of the acinus. VCAM-1 immune reactivity was diffuse but less intensive in the lobule. E-selectin expression was observed in sinusoidal cells attached to metastases. In tumour metastases the expression of ICAM-1, VCAM-1, and E-selectin was visible on the tumour vascular endothelium. Tumour infiltrating host cells sowing positive immunoreactivity for ICAM-1, VCAM-1, LFA-1, Mac-1, and VLA-4 were located mainly at the boundary between liver parenchyma and the metastasis. At the ultrastructural level, ICAM-1-positive immune deposits were observed on the cellular membrane and in some transport vesicles of gastric metastatic cells. Further, the expression of all adhesion molecules was confirmed to sinusoidal endothelial cells and tumour vessels. It is concluded that the enhanced expression of adhesion molecules in liver sinusoids could be a marker for the assessment of the ability of sinusoidal endothelial cells to control the recruitment of leukocytes and monocytes to the metastatic site. They could also direct the adhesion of new circulating tumour cells to sinusoidal endothelium.     

The inhibitory effect of alacepril,an angiotensin-converting enzyme inhibitor,on endothelial inflammatory response induced by oxysterol and TNF-α

Abstract

The objectives were to determine the effects of alacepril, an angiotensin-converting enzyme inhibitor, on the expression of adhesion molecules and monocyte adherence to endothelial cells induced by 7-ketocholesterol (7-KC) and tumor necrosis factor (TNF)-α. We used human aortic endothelial cells (HAECs) and U937 monocytic cells. Surface expression and mRNA levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) were determined by EIA and RT-PCR. Adherence of U937 to HAECs was assessed by adhesion assay. Incubation of HAEC with 7-KC increased the surface expression of protein and mRNA levels of ICAM-1 and VCAM-1 on HAECs and the production of reactive oxygen species (ROS) in HAECs. Pretreatment with alacepril reduced the enhanced expression of these molecules in a dose-dependent manner. The inhibitory effect of alacepril against 7-KC or TNF-α-induced CAMs expression was stronger than that of captopril or enalapril. Alacepril inhibited the production of ROS in HAECs stimulated by 7-KC or TNF-α. These results suggest that alacepril works as anti-atherogenic agent through inhibiting endothelial-dependent adhesive interactions with monocytes induced by 7-KC and TNF-α.     

Unique haplotype in exon 3 of cone opsin mRNA affects splicing of its precursor, leading to congenital color vision defect

Chemerin is a recently identified adipocytokine which plays a role on inflammation and adipocytes metabolism. However, its function in vasculature is largely unknown. We examined the effects of chemerin on vascular endothelial inflammatory states. Treatment of human umbilical vein endothelial cells with chemerin (300 ng/ml, 20 min) induced phosphorylation of Akt (Ser473) and endothelial nitric oxide (NO) synthase (eNOS) (Ser1177). Consistently, chemerin increased intracellular cyclic GMP content. Pretreatment with chemerin (1-300 ng/ml, 24 h) significantly inhibited phosphorylation of nuclear factor (NF)-κB p65 (Ser536) and p38 as well as vascular cell adhesion molecule (VCAM)-1 expression induced by tumor necrosis factor (TNF)-α (5 ng/ml, 20 min-6 h). Inhibitor of NF-κB or p38 significantly inhibited the TNF-α-induced VCAM-1 expression. Chemerin also inhibited TNF-α-induced VCAM-1 expression in rat isolated aorta. Moreover, chemerin significantly inhibited monocytes adhesion to TNF-α-stimulated endothelial cells. The inhibitory effect of chemerin on TNF-α-induced VCAM-1 was reversed by a NOS inhibitor. Conversely, an NO donor, sodium nitroprusside significantly inhibited TNF-α-induced VCAM-1. The present results for the first time demonstrate that chemerin plays anti-inflammatory roles by preventing TNF-α-induced VCAM-1 expression and monocytes adhesion in vascular endothelial cells. The effect is mediated via inhibiting activation of NF-κB and p38 through stimulation of Akt/eNOS signaling and NO production.     

Linoleic acid increases monocyte chemotaxis and adhesion to human aortic endothelial cells through protein kinase C- and cyclooxygenase-2-dependent mechanisms

The effects of polyunsaturated n-6 linoleic acid on monocyte-endothelial interactions were investigated with particular emphasis on the expression of platelet/endothelial cell adhesion molecule (PECAM)-1 and the role of protein kinase C (PKC) and cyclooxygenase-2 (COX-2). As a diet rich in polyunsaturated fatty acids may favour atherosclerosis in hyperglycaemia, this study was performed in both normal and high-glucose media using human aortic endothelial cells (HAEC). The HAEC were preincubated with normal (5 mM) or high (25 mM) D-glucose for 3 days before addition of fatty acids (0.2 mM) for 3 days. Linoleic acid enhanced PECAM-1 expression independently of tumor necrosis factor (TNF)-α and significantly increased TNF-α-induced monocyte adhesion to HAEC in comparison to the monounsaturated n-9 oleic acid. Chronic glucose treatment (25 mM, 6 days) did not modify the TNF-α-induced or fatty acid-induced changes in monocyte binding. The increase in monocyte binding was accompanied by a significant increase in E-selectin and vascular cell adhesion molecule (VCAM)-1 expression and could be abrogated by an interleukin (IL)-8 neutralising antibody and by the PKC and COX inhibitors. Inhibition of PKC-δ reduced VCAM-1 expression regardless of experimental condition and was accompanied by a significant decrease in monocyte binding. Conditioned medium from linoleic acid-treated HAEC grown in normal glucose conditions significantly increased THP-1 chemotaxis. These results suggest that linoleic acid-induced changes in monocyte chemotaxis and subsequent binding are not solely mediated by changes in adhesion molecule expression but may be due to secreted factors such as IL-8, monocyte chemoattractant protein-1 or prostaglandins (PGs) such as PGE(2), as IL-8 neutralisation and COX-2 inhibition reduced monocyte binding without changes in adhesion molecule expression.     

Peripheral benzodiazepine receptor regulates vascular endothelial activations via suppression of the voltage-dependent anion channel-1

Peripheral benzodiazepine receptor (PBR) is a multifunctional protein mainly found on the outer mitochondrial membrane. PBR expression is increased by tumor necrosis factor-α (TNF-α) in endothelial cells. Adenoviral overexpression of PBR inhibits monocyte adhesion, VCAM-1, and ICAM-1 expression in TNF-α-activated endothelial cells. Rotenone, cyclosporine A, and bongkrekic acid suppress TNF-α-induced VCAM-1 expression. Overexpression of PBR inhibits voltage-dependent anion channel-1 (VDAC-1) expression and the silencing of PBR increases VDAC-1 expression in endothelial cells. Moreover, TNF-α-induced VCAM-1 expression is suppressed by VDAC-1 gene silencing. PBR overexpression significantly decreases TNF-α-induced mitochondrial reactive oxygen species and MnSOD expression. These results suggest that PBR can inhibit endothelial activation and this action is related to the inhibition of mitochondrial ROS and/or VDAC-1 expression in endothelial cells.     

The anti-atherosclerotic effect of tanshinone IIA is associated with the inhibition of TNF-α-induced VCAM-1, ICAM-1 and CX3CL1 expression

Tanshinone IIA is one of the major diterpenes in Salvia miltiorrhiza. The inhibitory effect of Tanshinone IIA on atherosclerosis has been reported, but the underlying mechanism is not fully understood. The present study aimed to study the anti-atherosclerosis effect of Tanshinone IIA on the adhesion of monocytes to vascular endothelial cells and related mechanism. Results showed that Tanshinone IIA, at the concentrations without cytotoxic effect, dose-dependently inhibited the adhesion of THP-1 monocytes to the TNF-α-stimulated human vascular endothelial cells. The expressions of cell adhesion molecules including VCAM-1, ICAM-1 and E-selectin were induced by TNF-α in HUVECs at both the mRNA and protein levels. The mRNA and protein expressions of VCAM-1 and ICAM-1, but not E-selectin, were both significantly suppressed by Tanshinone IIA in a dose dependent manner. In addition, the TNF-α-induced mRNA expression of fractalkine/CX3CL1 and the level of soluble fractalkine were both reduced by Tanshinone IIA. We also found that Tanshinone IIA significantly inhibited TNF-α-induced nuclear translocation of NF-κB which was resulted from the inhibitory effect of Tanshinone IIA on the TNF-α-activated phosphorylation of IKKα, IKKβ, IκB and NF-κB. As one of the major components of Salvia miltiorrhiza, Tanshinone IIA alone exerted more potent effect on inhibiting the adhesion of monocytes to vascular endothelial cells when compared with Salvia miltiorrhiza. All together, these results demonstrate a novel underlying mechanism for the anti-inflammatory effect of Tanshinone IIA by modulating TNF-α-induced expression of VCAM-1, ICAM-1 and fractalkine through inhibition of TNF-α-induced activation of IKK/NF-κB signaling pathway in human vascular endothelial cells.     

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.     

Retinoid X receptor agonists impair arterial mononuclear cell recruitment through peroxisome proliferator-activated receptor-γ activation

Mononuclear cell migration into the vascular subendothelium constitutes an early event of the atherogenic process. Because the effect of retinoid X receptor (RXR)α on arterial mononuclear leukocyte recruitment is poorly understood, this study investigated whether RXR agonists can affect this response and the underlying mechanisms involved. Decreased RXRα expression was detected after 4 h stimulation of human umbilical arterial endothelial cells with TNF-α. Interestingly, under physiological flow conditions, TNF-α-induced endothelial adhesion of human mononuclear cells was concentration-dependently inhibited by preincubation of the human umbilical arterial endothelial cells with RXR agonists such as bexarotene or 9-cis-retinoid acid. RXR agonists also prevented TNF-α-induced VCAM-1 and ICAM-1 expression, as well as endothelial growth-related oncogene-α and MCP-1 release. Suppression of RXRα expression with a small interfering RNA abrogated these responses. Furthermore, inhibition of MAPKs and NF-κB pathways were involved in these events. RXR agonist-induced antileukocyte adhesive effects seemed to be mediated via RXRα/peroxisome proliferator-activated receptor (PPAR)γ interaction, since endothelial PPARγ silencing abolished their inhibitory responses. Furthermore, RXR agonists increased RXR/PPARγ interaction, and combinations of suboptimal concentrations of both nuclear receptor ligands inhibited TNF-α-induced mononuclear leukocyte arrest by 60-65%. In vivo, bexarotene dose-dependently inhibited TNF-α-induced leukocyte adhesion to the murine cremasteric arterioles and decreased VCAM-1 and ICAM-1 expression. Therefore, these results reveal that RXR agonists can inhibit the initial inflammatory response that precedes the atherogenic process by targeting different steps of the mononuclear recruitment cascade. Thus, RXR agonists may constitute a new therapeutic tool in the control of the inflammatory process associated with cardiovascular disease.