Inhibitory effects of clotrimazole on TNF-α-induced adhesion molecule expression and angiogenesis

Cell adhesion molecules play a pivotal role in chronic inflammation and pathological angiogenesis. In the present study, we investigated the inhibitory effects of clotrimazole (CLT) on tumor necrosis factor (TNF)-α-induced changes in adhesion molecule expression. CLT dose-dependently inhibited monocyte chemoattractant protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) expressions in TNF-α-stimulated HT29 colonic epithelial cells. This inhibitory action of CLT correlated with a significant reduction in TNF-α-induced adhesion of monocytes to HT29 cells, which was comparable to the inhibitory effects of anti-ICAM-1 and VCAM-1 monoclonal antibodies on monocyte-epithelial adhesion. These inhibitory actions of CLT were, at least in part, attributable to the inhibition of redox sensitive NF-κB activation, as CLT inhibited TNF-α-induced ROS generation as well as NF-κB nuclear translocation and activation in HT29 cells. Furthermore, the inhibition of TNF-α-induced monocyte adhesion was also mimicked by the specific NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC). Inflammatory mediators including TNF-α have known to promote angiogenesis, which in turn further contributes to inflammatory pathology. Therefore, we additionally evaluated whether CLT modulates TNF-α-induced angiogenesis using in vivo chick chorioallantoic membrane (CAM) assay. The CAM assay showed that CLT dose-dependently attenuated TNF-α-induced angiogenesis, and the effect was correlated with decreased inflammation of the CAM tissue. In conclusion, our results suggest that CLT can inhibit TNF-α-triggered expression of adhesion molecules, ICAM-1 and VCAM-1, and angiogenesis during inflammation.     

E-selectin is a viable route of infection for polymer-coated adenovirus retargeting in TNF-α-activated human umbilical vein endothelial cells

Background: E-selectin is an attractive endothelial cell surface marker in inflammation and cancer. Purpose: We sought to investigate retargeting of adenovirus via E-selectin as a viable pathway of infection in tumor necrosis factor-α (TNF-α)-activated human umbilical vein endothelial cells (HUVECs).

Methods: E1, E3-deleted Ad5 expressing cytomegalovirus immediate-early (CMV IE) promoter-driven luciferase (Adluc) was coated with an amino-reactive multivalent hydrophilic polymer based on poly [N-(2-hydroxypropyl) methacrylamide] to generate pHPMA-adenovirus (pcAdluc). This was then retargeted by covalent attachment of a mouse antihuman E-selectin monoclonal antibody (MHES mAb), purified from the H18/7 hybridoma cell line (MHESpcAdluc).

Results: MHESpcAdluc was efficiently taken up into HUVECs, generating a high level of transduction in TNF-α-treated E-selectin positive cells but not in untreated receptor-negative cells. Specific retargeting of MHESpcAdluc was demonstrated through reduced transduction of stimulated HUVEC when incubated in the presence of free E-selectin antibodies.

Discussion and conclusion: Our results suggest that E-selectin could be a valuable target for gene transfer strategies internalizing polymer-coated modified adenovirus particles through a viable receptor-mediated endocytosis pathway, generating adequate levels of transgene expression per virus genome copy without compromising the specific activity of the parental virus.     

3,4-oxo-isopropylidene-shikimic acid inhibits adhesion of polymorphonuclear leukocyte to TNF-α-induced endothelial cells in vitro

AIM：To examine the effect of 3,4-oxo-isopropylidene-shikimic acid (ISA) on human polymorphonuclear leukocyte (PMN) adhesion to human umbilical vein endothelial cells (HUVEC) and explore its mechanism. METHODS:Adhesion of PMN to HUVEC was measured by rose bengal staining assay. Cell-EL1SA and RT-PCR methods were used to examine the expression of adhesion molecules ICAM-1. Cell viability was detected with MTT assay.RESULTS: ISA (1-100μmol/L) effectively reduced PMN adhesion to TNF-α-induced HUVEC with the inhibitory rate from 17.2% to 53.5%, and exerted no effect on PMN adhesion to normal HUVEC. Adhesion molecule ICAM-1 surface protein and mRNA expression induced by TNF-α (400kU/L) were significantly inhibited by ISA. In addition, the cell viability of HUVEC was unchanged 48h after treatment with ISA. CONCLUSION: ISA inhibited TNF-α-stimulated PMN-HUVEC adhesion and expression of ICAM-1.     

Effects of tanshinone Ⅱ-A sullfonate on adhesion molecule expression of endothelial cells and platelets in vitro

探讨丹参酮ⅡＡ磺酸钠（Ｔａｎ）对培养人脐静脉内皮细胞（ＨＵＶＥＣ）和人血小板表达粘附分子的影响．方法：用流动血细胞计数仪测定肿瘤坏死因子（ＴＮＦα）诱导人脐静脉内皮细胞ＩＣＡＭ１和凝血酶诱导人血小板Ｐ选择素的表达．结果：ＨＵＶＥＣ经ＴＮＦα处理后，明显增加细胞表面ＩＣＡＭ１的表达，增加ＨＬ６０细胞粘附到内皮细胞表面达加入细胞总数的３０％±６％（对照组为４６％±０７％）．在ＴＮＦα处理前，用Ｔａｎ（２５－２００μｍｏｌ·Ｌ－１）与ＨＵＶＥＣ共孵育，则Ｔａｎ剂量依赖性地抑制ＴＮＦα的作用．Ｔａｎ（２５－２００μｍｏｌ·Ｌ－１）与人血小板孵育后，可剂量依赖性地抑制凝血酶诱导人血小板表面Ｐｓｅｌｅｃｔｉｎ的表达．结论：Ｔａｎ可抑制内皮细胞和血小板表达粘附分子．     

3,4-oxo-isopropylidene-shikimic acid inhibits adhesion of polymorphonuclear leukocyte to TNF-α-induced endothelial cells in υitro

INTRODUCTION Leukocyte infiltration to endothelium plays amajor role in the inflammatory response related tothrombosis, arteriosclerosis, and reperfusion injury, etc.Leukocyte infiltration requires a chain of reactions be-tween endothelial cells (EC) and leukocytes that initiallyretards intravascular leukocyte flow and finally leads toleukocyte transmigration through the endothelialmonolayer. These events are mediated by sequentialinteraction of different endothelial adhesion molecu…     

Modulation of Ecto-5′-Nucleotidase by phospholipids in human umbilical vein endothelial cells (HUVEC)

Ecto-5′-nucleotidase, the major enzyme controlling extracellular adenosine production, can be activated by phospholipids, e.g. lysophosphatidylcholine (LPC). This study examined the structural requirements of phospholipids to evoke this enzyme activation and figured out two new activators of ecto-5′-nucleotidase: platelet activating factor (PAF) and sphingosylphosphorylcholine (SPC). Potential signal transduction pathways including an involvement of protein kinase C and PAF-receptor were evaluated on the model of human umbilical vein endothelial cells (HUVEC). Cells were pre-incubated with 10 μM of various phospholipids including lysophosphatidylcholine, β-arachidonyl-γ-palmityl-α-phosphatidylcholine, β, γ-dipalmityl-α-phosphatidyl-choline, β,γ-dipalmityl-α-phosphatidylethanolamine, β,γ-dipalmityl-α-phosphatidylserine, γ-acyl-β-lyso-α-phosphatidylethanolamine, β-acetyl-γ-O-hexadecyl-α-phosphatidylcholine (platelet activating factor), lysophosphatidylic acid, sphingosine-1-phosphate and sphingosylphosphorylcholine. In the cell supernatant the extracellular dephosphorylation rate of the fluorescent AMP-analogue 1, N⁶-etheno-5′AMP to 1, N⁶-etheno-adenosine was measured by HPLC. Out of these ten structurally related phospholipids only lysophosphatidylcholine, sphingosylphosphatidylcholine and platelet activating factor dose-dependently increased the activity of ecto-5′-nucleotidase. Pharmacological blocking experiments revealed that neither the activation of PAF-receptor nor of protein kinase C were important for mediating the activation of ecto-5′-nucleotidase. Thus, using information on the known molecular structures of tested phospholipids, a phosphatidylcholine residue in α-position and a short chain length fatty acid esterified in β-position seem essential for activation of ecto-5′-nucleotidase by glycerophospholipids. Since all tested phospholipids have similar fatty acid chain lengths and residues in α-position, they should act similarly on membrane fluidity. It is concluded that the observed effects are not based on changes in membrane fluidity by the added phospholipids, but rather involve a yet to be determined phospholipid-receptor.     

Inhibition of TNF-α-induced adhesion molecule expression by diosgenin in mouse vascular smooth muscle cells via downregulation of the MAPK,Akt and NF-κB signaling pathways

Atherosclerosis is a chronic inflammatory disease and the expression of adhesion molecules on vascular smooth muscle cells (VSMCs) contributes to the progress of the disease. Diosgenin, a precursor of steroid hormones, has been shown to have a variety of biological activities including anti-inflammatory activity; however, its molecular mechanisms are poorly understood. This study examined the effect of diosgenin on the expression of adhesion molecules induced by TNF-α in cultured mouse VSMC cell line, MOVAS-1. Preincubation of VSMCs for 2 h with diosgenin (0.1–10 μM) dose-dependently inhibited TNF-α-induced adhesion of THP-1 monocytic cells and mRNA and protein expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Diosgenin abrogated TNF-α induced production of intracellular reactive oxygen species (ROS) and phosphorylation of p38, ERK, JNK and Akt. Diosgenin was also shown to inhibit NK-κB activation induced by TNF-α. Furthermore, diosgenin inhibited TNF-α-induced IκB kinase activation, subsequent degradation of IκBα, and nuclear translocation of NF-κB. Our results indicate that diosgenin inhibits the adhesive capacity of VSMC and the TNF-α-mediated induction of ICAM-1 and VCAM-1 in VSMC by inhibiting the MAPK/Akt/NF-κB signaling pathway and ROS production, which may explain the ability of diosgenin to suppress inflammation within the atherosclerotic lesion and modulate immune response.     

Inhibition of TNF-α-induced adhesion molecule expression by diosgenin in mouse vascular smooth muscle cells via downregulation of the MAPK, Akt and NF-κB signaling pathways

Atherosclerosis is a chronic inflammatory disease and the expression of adhesion molecules on vascular smooth muscle cells (VSMCs) contributes to the progress of the disease. Diosgenin, a precursor of steroid hormones, has been shown to have a variety of biological activities including anti-inflammatory activity; however, its molecular mechanisms are poorly understood. This study examined the effect of diosgenin on the expression of adhesion molecules induced by TNF-α in cultured mouse VSMC cell line, MOVAS-1. Preincubation of VSMCs for 2h with diosgenin (0.1-10 μM) dose-dependently inhibited TNF-α-induced adhesion of THP-1 monocytic cells and mRNA and protein expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Diosgenin abrogated TNF-α induced production of intracellular reactive oxygen species (ROS) and phosphorylation of p38, ERK, JNK and Akt. Diosgenin was also shown to inhibit NK-κB activation induced by TNF-α. Furthermore, diosgenin inhibited TNF-α-induced IκB kinase activation, subsequent degradation of IκBα, and nuclear translocation of NF-κB. Our results indicate that diosgenin inhibits the adhesive capacity of VSMC and the TNF-α-mediated induction of ICAM-1 and VCAM-1 in VSMC by inhibiting the MAPK/Akt/NF-κB signaling pathway and ROS production, which may explain the ability of diosgenin to suppress inflammation within the atherosclerotic lesion and modulate immune response.     

Tetrandrine inhibits Ca^2＋-activated chloride channel in cultured human umbilical vein endothelial cells

INTRODUCTION Tetrandrine (Tet, 6,6',7,12-tetramethoxy-2,2'- dim-ethyl-berbaman) is a purified bis-benzylisoquinoline al-kaloid derivedfrom the root ofa Chinese herb (Stephaniatetrandra S Moore)[1,2]. It was first shown as an anti-hypertensive agent in both normal and hypertensivesubjects in 1950s[3,4]. The primary anti-hypertensiveaction of Tet is presumably due to its vasodilatoryproperty, which was confirmed both in vivo (15 mg/kg in conscious rats) and in vitro (1-100 μmol/L,effecti…     

Proteomics analysis of human umbilical vein endothelial cells （HUVEC） after treatment with low molecular weight heparin

AIM: The endothelium is involved in the generation and the regulation of multiple physiological and pathological processes of blood vessels. Previously we confirmed low molecular weight heparin (LMWH) could inhibit tumor metastasis by protecting human umbilical vein endothelial cells (HUVEC). To understand the effects of LMWH on the protein expression of HUVEC, we performed a comprehensive proteomics to survey global changes in proteins after LMWH treatment in HUVEC cells. METHODS:     

Protective effects of ligustrazine on TNF-α-induced endothelial dysfunction

To investigate the effects of Ligustrazine, a compound derived from chuanxiong, on tumor necrosis factor-α (TNF-α) stimulated endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with TNF-α in vitro. Nitric oxide (NO) was measured as a standard of endothelial dysfunction. Two important indicators of autoimmunity, intracellular adhesion molecular-1 (ICAM-1) and heat shock protein 60 (HSP60), were selected to evaluate the influence of Ligustrazine on HUVECs. Ligustrazine (40 μg/ml) significantly reversed the decrease in NO production induced by TNF-α (5 ng/ml) in HUVECs. The expressions of ICAM-1 and HSP60 were increased by TNF-α treatment, but dramatically inhibited by treatment with ligustrazine in TNF-α-stimulated cells. Ligustrazine increased the production of NO in HUVECs and had an immunomodulatory effect on HUVECs stimulated with TNF-α by down-regulating the expression of ICAM-1 and HSP60. These results suggest that ligustrazine protects the endothelium via inhibition of immunological reactions, preventing atherosclerosis.     

Acanthoic acid inhibits LPS-induced inflammatory response by activating LXRα in human umbilical vein endothelial cells

Acanthoic acid, a pimaradiene diterpene isolated from Acanthopanax koreanum, has been reported to have anti-inflammatory activities. However, the effect of acanthoic acid on vascular inflammation has not been investigated. The aim of this study was to investigate the anti-inflammatory effects of acanthoic acid on lipopolysaccharide (LPS)-induced inflammatory response in human umbilical vein endothelial cells (HUVECs). The production of cytokines TNF-α and IL-8 was detected by ELISA. The expression of VCAM-1, ICAM-1, E-selectin, NF-κB and LXRα were detected by Western blotting. Adhesion of monocytes to HUVECs was detected by monocytic cell adhesion assay. The results showed that acanthoic acid dose-dependently inhibited LPS-induced TNF-α and IL-8 production. Acanthoic acid also inhibited TNF-α-induced IL-8 and IL-6 production. LPS-induced endothelial cell adhesion molecules, VCAM-1 and ICAM-1 were also inhibited by acanthoic acid. Acanthoic acid inhibited LPS-induced NF-κB activation. Furthermore, acanthoic acid dose-dependently up-regulated the expression of LXRα. In addition, our results showed that the anti-inflammatory effect of acanthoic acid was attenuated by transfection with LXRα siRNA. In conclusion, the anti-inflammatory effect of acanthoic acid is due to its ability to activate LXRα. Acanthoic acid may be a therapeutic agent for inflammatory cardiovascular disease.     

PPAR-γ agonists inhibit TGF-β1-induced chemokine expression in human tubular epithelial cells

Aim： Peroxisome proliferator-activated receptor-γ （PPAR-γ） has a wide range of biological functions, including anti-inflammation. In this study, we investigated the inhibitory effects of PPAR-γ on transforming growth factor β1 （TGF-β1）-induced interleukin-8 （IL-8） and monocyte chemoattractant protein-1 （MCP-1） expression in renal tubular epithelial cells （HK-2）.
Methods： HK-2 cells were pretreated with 15d-PGJ2 or troglitazone （TGL） and then treated with TGF-β1. Expression of MCP-1 and IL-8 was measured using real-time PCR and ELISA.
Results： Treatment with 5 ng/mL TGF-β1 for 24 h increased both MCP-1 and IL-8 mRNA and protein levels in HK-2 cells. Both 15d-PGJ2 at 2.5 and 5 12mol/L and TGL at 2.5 12mol/L exhibited inhibitory effects on TGF-β1-induced MCP-1 expression. Additionally, 15d-PGJ2 at 2.5 and 5 μmol/L and TGL at 2.5 μmol/L inhibited TGF-β1-induced expression of IL-8.
Conclusion： PPAR-γ agonists （15d-PGJ2 and TGL） could inhibit the TGF-β1-induced expression of chemokines in HK-2 cells. Our results suggest that PPAR-γ agonists have the potential to be used as a treatment regimen to reduce inflammation in renal tubulointerstitial disease.     

Withaferin A inhibits tumor necrosis factor-α-induced expression of cell adhesion molecules by inactivation of Akt and NF-κB in human pulmonary epithelial cells

We here investigated the functional effect of withaferin A on airway inflammation and its action mechanism. Withaferin A inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human lung epithelial A549 cells stimulated with tumor necrosis factor-α (TNF-α), resulting in the suppression of leukocyte adhesion to lung epithelial A549 cells. In addition, withaferin A inhibited TNF-α-induced expression of adhesion molecules (ICAM-1 and VCAM-1) protein and mRNA in a dose-dependent manner. Withaferin A prevented DNA binding activity of nuclear factor-κB (NF-κB) and nuclear translocation of NF-κB. It also inhibited phosphorylation of Akt and extracellular signal-regulated kinase (ERK), which are upstream in the regulation of adhesion molecules by TNF-α. Furthermore, withaferin A inhibited U937 monocyte adhesion to A549 cells stimulated by TNF-α, suggesting that it may inhibit the binding of these cells by regulating the expression of critical adhesion molecules by TNF-α. Taken together, these results suggest that withaferin A inhibits cell adhesion through inhibition of ICAM-1 and VCAM-1 expression, at least in part, by blocking Akt and down-regulating NF-κB activity.     

Sargachromenol protects against vascular inflammation by preventing TNF-α-induced monocyte adhesion to primary endothelial cells via inhibition of NF-κB activation

Vascular inflammation is a key factor in the pathogenesis of atherosclerosis. The purpose of this study was to investigate the protective effects of sargachromenol (SCM) against tumor necrosis factor (TNF)-α-induced vascular inflammation. SCM decreased the expression of cell adhesion molecules, including intracellular adhesion molecule-1 and vascular cell adhesion molecule-1, in TNF-α-stimulated human umbilical vein endothelial cells (HUVECs), resulted in reduced adhesion of monocytes to HUVECs. SCM also decreased the production of monocyte chemoattractant protein-1 and matrix metalloproteinase-9 in TNF-α-induced HUVECs. Additionally, SCM inhibited activation of nuclear factor kappa B (NF-κB) induced by TNF-α through preventing the degradation of inhibitor kappa B. Moreover, SCM reduced the production of reactive oxygen species in TNF-α-treated HUVECs. Overall, SCM alleviated vascular inflammation through the regulation of NF-κB activation and through its intrinsic antioxidant activity in TNF-α-induced HUVECs. These results indicate that SCM may have potential application as a therapeutic agent against vascular inflammation.