Adhesion Of HL-60 Cells Research Articles

Luteolin Inhibits Indoxyl Sulfate-Induced ICAM-1 and MCP-1 Expression by Inducing HO-1 Expression in EA.hy926 Human Endothelial Cells.

In patients with chronic kidney disease, the uremic toxin indoxyl sulfate (IS) accelerates kidney damage and the progression of cardiovascular disease. IS may contribute to vascular diseases by inducing inflammation in endothelial cells. Luteolin has documented antioxidant and anti-inflammatory properties. This study aimed to investigate the effect of luteolin on IS-mediated reactive oxygen species (ROS) production and intercellular adhesion molecule (ICAM-1) and monocyte chemoattractant protein (MCP-1) expression in EA.hy926 cells and the possible mechanisms involved. IS significantly induced ROS production (by 6.03-fold, p < 0.05), ICAM-1 (by 2.19-fold, p < 0.05) and MCP-1 protein expression (by 2.18-fold, p < 0.05), and HL-60 cell adhesion (by 31%, p < 0.05), whereas, luteolin significantly decreased IS-induced ROS production, ICAM-1 and MCP-1 protein expression, and HL-60 cell adhesion. Moreover, luteolin attenuated IS-induced nuclear accumulation of p65 and c-jun. Luteolin dose-dependently increased heme oxygenase-1 (HO-1) expression and the maximum fold induction of HO-1 by luteolin was 3.68-fold (p < 0.05), whereas, HO-1 knockdown abolished the suppression of ICAM-1 and MCP-1 expression by luteolin. Luteolin may protect against IS-induced vessel damage by inducing HO-1 expression in vascular endothelial cells, which suppresses nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1) mediated ICAM-1 and MCP-1 expression.

Portulacerebroside a Inhibits Adhesion, Migration, and Invasion of Human Leukemia HL60 Cells and U937 Cells through the Regulation of p38/JNK Signaling Pathway [Retraction

[This retracts the article DOI: 10.2147/OTT.S117523.].

Inhibitory Effect of Ethanol Extract from Folium et Ramulus Securinegae on TNF‐α induced vascular inflammation in Human Umbilical Vein Endothelial Cells

Folium et Ramulus Securinegae (FRS) is known as a drug that protects and strengthen kidney and spleen by improving the blood circulation and relaxing muscles and tendons. This study investigated whether FRS has protective effect of inhibiting the vascular inflammation in human umbilical vein endothelial cells (HUVECs) by inducing nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) coupling pathway. Results showed that FRS suppressed TNF-α (Tumor necrosis factor–α) induced protein and mRNA levels of cell adhesion molecules such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and Interleukin-6 (IL-6). Pretreatment of HUVEC with FRS decreased the adhesion of HL-60 cells to Ox-LDL (Oxidized Low-Density-Lipoprotein)-induced HUVEC. Moreover, FRS inhibited TNF-α induced intracellular reactive oxygen species (ROS) production. FRS also inhibited phosphorylation of IκB-α in cytoplasm and translocation of NF-κB (Nuclear factor-kappa B) p65 to the nucleus. FRS increased NO production, as well increased the phosphorylation of eNOS and Akt (protein kinase B) which are related with NO production. In addition, FRS increased the protein expression of GTPCH (Guanosine triphosphate cyclohydrolase Ⅰ) and the production of BH4 in HUVEC which are related with eNOS coupling pathway. Overall, the present data suggest that FRS has a protective effect against vascular inflammation and can be a potential therapeutic agent for early atherosclerosis.

Human Immunodeficiency Virus-1–Tat Protein Induces the Cell Surface Expression of Endothelial Leukocyte Adhesion Molecule-1, Vascular Cell Adhesion Molecule-1, and Intercellular Adhesion Molecule-1 in Human Endothelial Cells

AbstractHuman vascular endothelial cells (EC) have been implicated in the dissemination of human immunodeficiency virus type-1 (HIV-1). HIV-1–tat, a viral gene product essential for HIV replication, has been shown to interact with different cell types, altering their growth and inducing gene expression. In the present report, we have examined the effect of HIV-tat on the expression of various adhesion molecules in human umbilical vein EC. Our results show that treatment of EC with HIV-tat induces the cell surface expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and endothelial leukocyte adhesion molecule-1 in a timeand dose-dependent manner. Cycloheximide abolished the HIV-tat–dependent induction of all the adhesion molecules, indicating that protein synthesis was required for induction. The effect of HIV-tat on expression of adhesion molecules was potentiated by tumor necrosis factor (TNF ), a well-known inducer of adhesion molecules. Like TNF, HIV-tat also enhanced the adhesion of human promyelomonocytic HL-60 cells to EC, and this effect was abolished by treatment with antibodies either against HIV-tat or adhesion molecules. Our results thus indicate that the HIV-tat protein can activate human vascular EC to induce the expression of various adhesion molecules that may play a role in the extravasation of HIV-infected cells.

Anti-Vascular Inflammatory Effect of Ethanol Extract from Securinega suffruticosa in Human Umbilical Vein Endothelial Cells.

Securiniga suffruticosa is known as a drug that has the effect of improving the blood circulation and relaxing muscles and tendons, thereby protects and strengthen kidney and spleen. Therefore, in this study, treatment of Securiniga suffruticosa showed protective effect of inhibiting the vascular inflammation in human umbilical vein endothelial cells (HUVECs) by inducing nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) coupling pathway. In this study, Securiniga suffruticosa suppressed TNF-α (Tumor necrosis factor–α) induced protein and mRNA levels of cell adhesion molecules such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and Interleukin-6 (IL-6). Pretreatment of HUVEC with Securiniga suffruticosa decreased the adhesion of HL-60 cells to Ox-LDL (Oxidized Low-Density-Lipoprotein)-induced HUVEC. Moreover, Securiniga suffruticosa inhibited TNF-α induced intracellular reactive oxygen species (ROS) production. Securiniga suffruticosa also inhibited phosphorylation of IκB-α in cytoplasm and translocation of NF-κB (Nuclear factor-kappa B) p65 to the nucleus. Securiniga suffruticosa increased NO production, as well increased the phosphorylation of eNOS and Akt (protein kinase B) which are related with NO production. In addition, Securiniga suffruticosa increased the protein expression of GTPCH (Guanosine triphosphate cyclohydrolase Ⅰ) and the production of BH4 in HUVEC which are related with eNOS coupling pathway. In conclusion, Securiniga suffruticosa has a protective effect against vascular inflammation and can be a potential therapeutic agent for early atherosclerosis.

Characterization of a P-selectin-binding moiety from Bupleurum chinense polysaccharide and its antagonistic effect against P-selectin-mediated function

P-selectin is a promising therapeutic target for acute inflammation-related diseases, and interest has been growing in the search for high-affinity glycoconjugate ligands that can target the initial P-selectin-mediated recruitment of neutrophils to the site of inflammation. In our previous study, we isolated a water-soluble polysaccharide (BCPS) from Bupleurum chinense and showed that it exhibits anti-inflammatory effect by antagonizing P-selectin-mediated adhesion of HL-60 cells to CHO-P cells. In this study, we prepared a P-selectin-based affinity chromatography medium and used it to purify the P-selectin-binding moiety of BCPS. The purified P-selectin-binding moiety of BCPS, designated as BCPS-m, was mainly composed of arabinose, galactose and glucose, and had a relative molecular weight of 3600 Da. The backbone of BCPS-m was composed of 1,5-linked arabinose, 1,4-linked and 1,4,6-linked glucose, and with branched 1-linked glucose or galactose terminal. BCPS-m could disrupt the P-selectin-mediated binding of HL-60 cells to CHO-P cells (CHO cells that stably expressed an exogenous P-selectin). It also blocked the interaction between P-selectin and its physiological ligand PSGL-1 significantly, resulting in much greater reduction (77%) in P-selectin-PSGL-1 binding than that caused by BCPS (35%). The data suggested that BCPS-m could be the key P-selectin-binding moiety of BCPS, and that it may be a better P-selectin antagonist than BCPS.

PPARγ Agonists Inhibit Adhesion Signal to Endothelial Cells in the Differentiation Induction of HL-60 Acute Promyelocytic Leukemia Cells

All-trans retinoic acid (ATRA) has successfully been used in the treatment of acute promyelocytic leukemia (APL) patients, with a remission rate of greater than 90% . Despite the high cure rates, induction mortality is a still a problem in APL. One of the most common causes of death was the differentiation syndrome (DS) . The early administration of high-dose dexamethasone at the onset of the first signs or symptoms of DS is crucial, however specific biological therapies to counteract the syndrome are still not available. Peroxisome proliferator activated receptor gamma (PPARγ) is a ligand-dependent transcription factor and a member of the nuclear receptor superfamily, which is expressed in normal monocytes, various leukemias, and epithelial malignancies. PPARγ is highly induced in differentiating myeloid cells and subsequently contributes to their differentiation. Differentiation induction of APL cells is associated with increased expression of specific adhesion molecules and inflammatory cytokines, which may promote activation, migration, and adhesion of these cells. Here, we studied the effect of PPARγ agonists on the adhesion of a human leukemia cell line (HL-60) to endothelial cells. HL-60 cells were differentiated into macrophage-like cells by a PKC activator, 12-O-Tetradecanoylphorbol-13-acetate (TPA). Differentiation was determined by an increase in reactivity with the CD11b antibody. For the adhesion assay, the Matrigel transwell system was used. During the differentiation of HL-60 cells, PPARγ agonists acitvate TPA-induced CD11b expression. However, PPARγ agonists completely blocked TPA-induced ICAM-1 expression of endothelial cells, which resulted in the inhibition of adhesion of HL-60 cells to endothelial cells. These responses also were reversed by PPARγ antagonist (GW9662), indicating that PPARγ agonists inhibits the adhesion of the HL-60 cells to endothelial cells through a PPARγ dependent mechanism. These results suggest that PPARγ agonists inhibit TPA-induced adhesion signal in the between HL-60 cells and endothelial cells, and may control differentiation syndrome in APL patients. DisclosuresNo relevant conflicts of interest to declare.

Portulacerebroside A inhibits adhesion, migration, and invasion of human leukemia HL60 cells and U937 cells through the regulation of p38/JNK signaling pathway.

Acute myeloid leukemia (AML) is a highly malignant hematopoietic tumor. This study aimed to explore the effect of portulacerebroside A (PCA) on the adhesion, migration, and invasion in human leukemia HL60 cells and U937 cells and clarify the possible mechanisms involved, which could provide potential strategies for the treatment of AML. By methyl thiazolyl tetrazolium analysis, it was found that PCA (1–10 μM) suppressed the cell viability in a time- and dose-dependent manner. A total of 1, 2, and 5 μM of PCA dramatically inhibited the adhesion, migration, and invasion of HL60 cells and U937 cells in a dose-dependent manner. Phosphorylation level of JNK and P38 protein level was measured by Western blot. After the real-time quantification polymerase chain reaction and Western blot detection of the total RNA and protein, messenger RNA, and protein expression levels of Ras homologous C (RhoC), metastasis-associated gene 1 (MTA1) and matrix metalloproteinase-2/9 (MMP-2/9) were decreased significantly in a dose-dependent manner. The phosphorylation level of c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinase (P38) was decreased dramatically in HL60 cells and U937 cells after PCA treatment. In conclusion, PCA significantly inhibits the adhesion, migration, and invasion of HL60 cells and U937 cells by suppressing the p38/JNK pathway and regulating the expressions of related genes.

Granulocyte colony-stimulating factor inhibits CXCR4/SDF-1α signaling and overcomes stromal-mediated drug resistance in the HL-60 cell line.

Combining cytarabine, aclarubicin and granulocyte colony-stimulating factor (G-CSF) has demonstrated marked efficacy in the treatment of elderly and relapsed/refractory patients with acute myeloid leukemia (AML); however, the role of G-CSF remains poorly understood. The present study aimed to investigate the ability of G-CSF to overcome stromal-mediated drug resistance and the underlying molecular mechanism. Two types of co-culture models were established in the HS-5 human bone marrow/stromal and HL-60 human promyelocytic leukemia cell lines, in order to imitate the interactions between stromal and leukemia cells in vitro, which is mediated by the stromal cell-derived factor (SDF)-1α signaling axis. In the present study, HL-60 cells were attracted and adhered to HS-5 cells using migration assay and flow cytometry, respectively; however, these interactions were inhibited by treatment with G-CSF and/or the C-X-C chemokine receptor type 4 (CXCR4) antagonist, AMD3100. Co-culture with HS-5 cells, including direct and indirect contact, protected HL-60 cells against spontaneous apoptosis or drug-induced apoptosis; however, these protective effects were disrupted by treatment with G-CSF and/or AMD3100. Notably, G-CSF and/or AMD3100 did not alter cell viability or apoptosis when HL-60 cells were cultured with medium alone. In addition, G-CSF significantly reduced the expression levels of surface CXCR4 protein, total CXCR4 protein and CXCR4 mRNA, and significantly upregulated the expression of microRNA (miR)-146a. Conversely, AMD3100 significantly reduced surface CXCR4 expression levels, but not the total CXCR4, CXCR4 mRNA or miR-146a expression levels. The results of the present study suggested that interfering with the CXCR4/SDF-1α signaling axis via G-CSF inhibited the migration and adhesion of HL-60 cells to HS-5 cells and eliminated HS5 cell-mediated protective effects. Furthermore, G-CSF administration reduced CXCR4 expression levels by upregulating the expression of miR-146a, whereas AMD3100 appeared to be predominantly dependent on receptor internalization. Therefore, a G-CSF/miR-146a/CXCR4 pathway may explain how G-CSF inhibits CXCR4/SDF-1α signaling and overcomes stromal cell-mediated drug resistance in acute myeloid leukemia.

Clopidogrel Protects Endothelium by Hindering TNFα-Induced VCAM-1 Expression through CaMKKβ/AMPK/Nrf2 Pathway

Clopidogrel (INN), an oral antiplatelet drug, has been revealed to have a number of biological properties, for instance, anti-inflammation and antioxidation. Oxidative stress plays an imperative role in inflammation, diabetes mellitus, atherosclerosis, and cancer. In the present study, human aortic endothelial cells (HAECs) were employed to explore the anti-inflammatory activity of INN. INN reduced TNFα-induced reactive oxygen species (ROS) generation and time-dependently prompted the expression and activity of heme oxygenase 1 (HO-1). Cellular glutathione (GSH) levels were augmented by INN. shHO-1 blocked the INN suppression of TNFα-induced HL-60 cell adhesion. The CaMKKβ/AMPK pathway and Nrf2 transcriptional factor were implicated in the induction of HO-1 by INN. Additionally, TNFα dramatically augmented VCAM-1 expression at protein and mRNA levels. INN treatment strikingly repressed TNFα-induced expression of VCAM-1 and HL-60 cell adhesion. Compound C, an AMPK inhibitor, and shNrf2 abolished TNFα-induced expression of VCAM-1 and HL-60 cell adhesion. Our data suggest that INN diminishes TNFα-stimulated VCAM-1 expression at least in part via HO-1 induction, which is CaMKKβ/AMPK pathway-dependent.

Docosahexaenoic acid inhibits inflammation via free fatty acid receptor FFA4, disruption of TAB2 interaction with TAK1/TAB1 and downregulation of ERK-dependent Egr-1 expression in EA.hy926 cells.

Inflammation is intimately associated with many cardiovascular events and docosahexaenoic acid (DHA) has been shown to protect against CVD. Egr-1 has emerged as a key regulator in the development of atherosclerosis. Free fatty acid receptor 4 (FFA4) is an n-3 FA membrane receptor. Tumor necrosis factor alpha (TNF-α) is an inflammatory mediator and transforming growth factor-β-activated kinase 1 (TAK1) is essential in the TNF-α-mediated activation of NF-κB. We examined the mechanisms underlying DHA inhibition of inflammation in human EA.hy926 cells. TNF-α markedly induced the interaction between TAK1 binding protein (TAB) 2 and TAK1/TAB1, the phosphorylation of ERK, p38 MAPK and Akt, the expression of Egr-1 and ICAM-1, and HL-60 (monocyte-like) cell adhesion. Pretreatment with DHA attenuated TNF-α-induced phosphorylation of ERK, expression of Egr-1 and ICAM-1 and HL-60 cell adhesion. Transfection with siFFA4 reversed the DHA-mediated inhibition of TNF-α-induced Egr-1 and ICAM-1 expression, HL-60 cell adhesion and NF-κB and DNA-binding activity. Our results suggest that the anti-inflammatory effect of DHA on the endothelium is at least partially linked to FFA4, disruption of TAB2 interaction with TAK1/TAB1 and downregulation of ERK-dependent Egr-1 and ICAM-1 expression, which leads to less HL-60 cell adhesion to TNF-α-stimulated EA.hy926 cells.

Effects of Simulated Microgravity on Functions of Neutrophil-like HL-60 Cells

Altered gravity, especially microgravity affects cellular functions of immune cells and can result in immune dysfunction for long-term, manned spaceflight and space exploration. The underlying mechanism, however, of sensing and responding to the gravity alteration is poorly understood. Here, a rotary cell culture system (RCCS) bioreactor was used to elucidate the effects of simulated microgravity on polymorphonuclear neutrophils (PMN)-like HL-60 cells. Alteration of cell morphology, up-regulation of (nitric oxide) NO production, enhancement of interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemotactic protein 1 (MCP-1) secretion, and diversity of cellular adhesion molecule expression were observed for the cells cultured in RCCS, leading to the up-regulated inflammatory immune responses and host defense. It was also indicated that such alterations in biological responses of PMNs mediated the reduced rolling velocity and decreased adhesion of PMN-like HL-60 cells on endothelial cells under shear flow. This work furthers the understandings in the effects and mechanism of microgravity on PMN functions, which are potentially helpful for optimizing the countermeasures to immune suppression in the future long-term, manned spaceflight.

A Convenient Technique to Fix Suspension Cells on a Coverslip for Microscopy.

Human myeloid HL-60 cells are usually cultured in suspension in medium containing 5% to 10% fetal bovine serum (FBS) and thus are often difficult to adhere to a coverslip. In this unit, we describe how removal of FBS from the culture medium facilitates adhesion of HL-60 cells to coverslips. Importantly, HL-60 cells that adhere to the coverslips immersed in FBS-free medium can be immobilized in situ by conventional chemical fixatives and thus permeabilized for probing cellular structures using specific dyes and/or reagents, followed by microscopic observation. All-trans-retinoic-acid-exposed differentiated HL-60 cells, which have properties similar to neutrophils, can also adhere efficiently to coverslips in FBS-free medium. Because the procedure is not complex and special equipment is not required, the simplicity and cost effectiveness of this FBS-free cell adhesion protocol may be beneficial to researchers who are interested in assessing the structure and function of suspension cells using microscopy.

Andrographolide inhibits TNFα-induced ICAM-1 expression via suppression of NADPH oxidase activation and induction of HO-1 and GCLM expression through the PI3K/Akt/Nrf2 and PI3K/Akt/AP-1 pathways in human endothelial cells

Andrographolide, the major bioactive component of Andrographis paniculata, has been demonstrated to have various biological properties including anti-inflammation, antioxidation, and anti-hepatotoxicity. Oxidative stress is considered a major risk factor in aging, inflammation, cancer, atherosclerosis, and diabetes mellitus. NADPH oxidase is a major source of endogenous reactive oxygen species (ROS). In this study, we used EA.hy926 endothelial-like cells to explore the anti-inflammatory activity of andrographolide. Andrographolide attenuated TNFα-induced ROS generation, Src phosphorylation, membrane translocation of the NADPH oxidase subunits p47phox and p67phox, and ICAM-1 gene expression. In the small hairpin RNA interference assay, shp47phox abolished TNFα-induced p65 nuclear translocation, ICAM-1 gene expression, and adhesion of HL-60 cells. Andrographolide induced the gene expression of heme oxygenase 1 (HO-1) and glutamate cysteine ligase modifier subunit (GCLM) in a time-dependent manner. Cellular glutathione (GSH) content was increased by andrographolide. shGCLM attenuated the andrographolide-induced increase in GSH content and reversed the andrographolide inhibition of HL-60 adhesion. shHO-1 showed a similar effect on andrographolide inhibition of HL-60 adhesion to shGCLM. The mechanism underlying the up-regulation of HO-1 and GCLM by andrographolide was dependent on the PI3K/Akt pathway, and both the Nrf2 and AP-1 transcriptional factors were involved. Our results suggest that andrographolide attenuates TNFα-induced ICAM-1 expression at least partially through suppression of NADPH oxidase activation and induction of HO-1 and GCLM expression, which is PI3K/Akt pathway-dependent.

Telmisartan inhibits the proinflammatory effects of homocysteine on human endothelial cells through activation of the peroxisome proliferator-activated receptor-δ pathway.

The aim of this study was to investigate the inhibition capacity of telmisartan to endothelial inflammation induced by homocysteine (Hcy) and discuss the proposed mechanism invitro. Human umbilical vein endothelial cells (HUVECs) were prepared by collagenase digestion and cultured invitro. An increase in monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1) was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) as markers of Hcy-induced endothelial inflammation. HL-60 cell adhesion to HUVECs was measured by rose bengal staining. Nuclear, cytosolic and total nuclear transcription factor-κB (NF-κB) p65 levels were analyzed by western blotting. Peroxisome proliferator-activated receptor-δ (PPARδ) expression by HUVECs exposed to Hcy with or without telmisartan pretreatment was analyzed by RT-PCR and western blotting. Hcy significantly increased the levels of MCP-1 mRNA, VCAM-1 mRNA and monocyte binding to HUVECs. These effects were significantly attenuated by pretreatment with telmisartan and PPARδ agonists. The effect of telmisartan was inhibited by PPARδ antagonists. The Hcy-mediated downregulation of PPARδ mRNA and protein of HUVECs was inhibited by telmisartan. Hcy-mediated upregulation of NF-κB p65 protein levels in nuclear extracts was inhibited by telmisartan and PPARδ agonists. In conclusion, telmisartan exerts potent anti-inflammatory effects in endothelial cells, probably via a binary mechanism involving PPARδ activation and inhibition of the nuclear translocation of NF-κB.

Lipid Raft Is Required for PSGL-1 Ligation Induced HL-60 Cell Adhesion on ICAM-1

P-selectin glycoprotein ligand-1 (PSGL-1) and integrins are adhesion molecules that play critical roles in host defense and innate immunity. PSGL-1 mediates leukocyte rolling and primes leukocytes for integrin-mediated adhesion. However, the mechanism that PSGL-1 as a rolling receptor in regulating integrin activation has not been well characterized. Here, we investigate the function of lipid raft in regulating PSGL-1 induced β2 integrin-mediated HL-60 cells adhesion. PSGL-1 ligation with antibody enhances the β2 integrin activation and β2 integrin-dependent adhesion to ICAM-1. Importantly, with the treatment of methyl-β-cyclodextrin (MβCD), we confirm the role of lipid raft in regulating the activation of β2 integrin. Furthermore, we find that the protein level of PSGL-1 decreased in raft fractions in MβCD treated cells. PSGL-1 ligation induces the recruitment of spleen tyrosine kinase (Syk), a tyrosine kinase and Vav1 (the pivotal downstream effector of Syk signaling pathway involved in cytoskeleton regulation) to lipid raft. Inhibition of Syk activity with pharmacologic inhibitor strongly reduces HL-60 cells adhesion, implicating Syk is crucial for PSGL-1 mediated β2 integrin activation. Taken together, we report that ligation of PSGL-1 on HL-60 cells activates β2 integrin, for which lipid raft integrity and Syk activation are responsible. These findings have shed new light on the mechanisms that connect leukocyte initial rolling with subsequent adhesion.

Anti-TNF-α Activity of Portulaca oleracea in Vascular Endothelial Cells

Vascular inflammation plays a key role in the pathogenesis and progression of atherosclerosis, a main complication of diabetes. The present study investigated whether an aqueous extract of Portulaca oleracea (AP) prevents the TNF-α-induced vascular inflammatory process in the human umbilical vein endothelial cell (HUVEC). The stimulation of TNF-α induced overexpression of adhesion molecules affects vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1 and E-selectin for example. However, AP significantly suppressed TNF-α-induced over-expression of these adhesion molecules in a dose-dependent manner. In addition, pretreatment with AP dose-dependently reduced an increase of the adhesion of HL-60 cells to TNF-α-induced HUVEC. Furthermore, we observed that stimulation of TNF-α significantly increased intracellular reactive oxygen species (ROS) production. However, pretreatment with AP markedly blocked TNF-α-induced ROS production in a dose-dependent manner. The western blot and immunofluorescence analysis showed that AP inhibited the translocation of p65 NF-κB to the nucleus. In addition, AP suppressed the TNF-α-induced degradation of IκB-α and attenuated the TNF-α-induced NF-κB binding. AP also effectively reduced TNF-α-induced mRNA expressions of monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-8 in a dose-dependent manner. Taken together, AP prevents the vascular inflammatory process through the inhibition of intracellular ROS production and NF-κB activation as well as the reduction of adhesion molecule expression in TNF-α-induced HUVEC. These results suggested that AP might have a potential therapeutic effect by inhibiting the vascular inflammation process in vascular diseases such as atherosclerosis.

THE BIOMECHANICS OF DRUG-TREATED LEUKEMIA CELLS INVESTIGATED USING OPTICAL TWEEZERS

Leukemia is a very common cancer worldwide, and different drugs have been applied to treat the disease. However, the influence of the drugs on the biomechanical properties of leukemia cells, which are related to the risk of leukostasis, is still unknown. Moreover, accurate measurement of biomechanical properties of leukemia cells is still a challenging task because of their non-adherent nature and high sensitivity to the surrounding physiological conditions. In this study, a protocol to measure the biomechanical properties of leukemia cells by performing indentation tests using optical tweezers is proposed. The biomechanical properties of normal leukemia cells and cells treated with various cancer drugs, including phorbol 12-myristate 13-acetate (PMA), all-trans retinoic acid (ATRa), Cytoxan (CTX), and Dexamethasone (DEX), were measured. The adhesion between the cells and certain proteins existing in the extracellular matrix, i.e., fibronetin and collagen I, was also characterized with the help of a static adhesion assay. It was found that after treatment by ATRa, CTX, and DEX, the cells became softer, and the adhesion between the cells and the proteins became weaker. PMA treatment caused no change in the stiffness of the HL60 cells, but increased the stiffness of the K562 cells, and increased the cell–protein adhesion of both K562 cells and HL60 cells.

Suppression of endothelial cell activity by inhibition of TNFα

IntroductionTNFα is a proinflammatory cytokine that plays a central role in the pathogenesis of rheumatoid arthritis (RA). We investigated the effects of certolizumab pegol, a TNFα blocker, on endothelial cell function and angiogenesis.MethodsHuman dermal microvascular endothelial cells (HMVECs) were stimulated with TNFα with or without certolizumab pegol. TNFα-induced adhesion molecule expression and angiogenic chemokine secretion were measured by cell surface ELISA and angiogenic chemokine ELISA, respectively. We also examined the effect of certolizumab pegol on TNFα-induced myeloid human promyelocytic leukemia (HL-60) cell adhesion to HMVECs, as well as blood vessels in RA synovial tissue using the Stamper-Woodruff assay. Lastly, we performed HMVEC chemotaxis, and tube formation.ResultsCertolizumab pegol significantly blocked TNFα-induced HMVEC cell surface angiogenic E-selectin, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression and angiogenic chemokine secretion (P < 0.05). We found that certolizumab pegol significantly inhibited TNFα-induced HL-60 cell adhesion to HMVECs (P < 0.05), and blocked HL-60 cell adhesion to RA synovial tissue vasculature (P < 0.05). TNFα also enhanced HMVEC chemotaxis compared with the negative control group (P < 0.05) and this chemotactic response was significantly reduced by certolizumab pegol (P < 0.05). Certolizumab pegol inhibited TNFα-induced HMVEC tube formation on Matrigel (P < 0.05).ConclusionOur data support the hypothesis that certolizumab pegol inhibits TNFα-dependent leukocyte adhesion and angiogenesis, probably via inhibition of angiogenic adhesion molecule expression and angiogenic chemokine secretion.

Soluble LR11/SorLA, a Potential Circulating Marker Indicating the G-CSF-Induced Mobilization of Hematopoietic Stem Cells, Is a Modulator of G-CSF-Mediated Migration of HL-60 Cells,

Abstract 3210 Introduction:LR11 (also called SorLA or SORL1) is a type I membrane protein from which a large extracellular part, soluble LR11 (sLR11), is released by proteolytic shedding. LR11 plays a key role in the migration of undifferentiated vascular smooth muscle cells. Circulating sLR11 levels are a marker of accumulation of immature vascular cells, with immature human CD34+CD38− hematopoietic precursor cells having been shown to express high levels of LR11 mRNA. Recently, we showed that LR11 is specifically and highly expressed on the surface of acute leukemia cells in addition to normal leukocytes (unpublished data). Granulocyte-colony stimulating factor (G-CSF) stimulates hematopoietic stem cell (HSC) mobilization from the bone marrow (BM) to peripheral circulation and is commonly accepted as inducer of HSC mobilization in patients with hematological diseases. However, the mechanism underlying G-CSF-mediated release of HSCs from BM into peripheral circulation has not been fully elucidated. This study examined serum sLR11 levels during collection of HSCs and the role of sLR11 in migration and adhesion of HL-60 cells. Materials and methods:We analyzed serum samples of 15 patients who underwent the HSC collection procedure at Chiba University Hospital between 2010 and 2011. We initially examined the effects of G-CSF on the release of sLR11 from HL-60 cells into culture supernatants by an in vitro assay. We then measured expression of LR11 on HL-60 cells by flow cytometric analysis. sLR11 levels were measured in serum or culture supernatants by an ELISA method. A migration assay was performed using HL-60 cells incubated with or without G-CSF in transwell chambers with or without HUVECs, which were pre-incubated with or without TNF-a. An adhesion assay was performed using fibronectin-coated plates with or without HUVECs pre-incubated with or without TNF-a. Results:Circulating sLR11 levels were associated with time-related changes in the number of peripheral leukocytes during collection of HSCs following G-CSF treatment. Figure 1 shows the time course of two representative cases. In Case 1, a patient with malignant lymphoma, the number of leukocytes increased concurrently with an increase in serum sLR11 levels after chemotherapy, and then both decreased simultaneously. Case 2 was a patient with POEMS syndrome who was treated with G-CSF at the time of stem cell harvest. Similar to Case 1, following administration of G-CSF, the number of leukocytes increased concurrently with the increase in serum sLR11 levels, and then both decreased simultaneously. A close relationship was observed between serum sLR11 levels and leukocyte count, with a relative ratio of 0.63. We examined sLR11 in BM plasma by ELISA and showed that sLR11 was abundant in BM and was produced by BM hematological cells. sLR11 levels were increased significantly in the media containing HL-60 cells incubated for 12 h with 100 ng/ml G-CSF compared with those containing HL-60 cells incubated without G-CSF. Surface expression of LR11 on HL-60 cells increased with G-CSF treatment compared with controls (Figure 2), but not with SDF-1 treatment. In the migration assay, sLR11 stimulated the migration of HL-60 cells in a dose-dependent manner. Stimulation of migration activity by sLR11 was observed clearly in the presence of G-CSF with or without HUVECs. In the adhesion assay, sLR11 induced attachment of HL-60 cells to fibronectin-coated plates or HUVECs. This attachment increased when HL-60 cells were incubated with G-CSF. In a trans-endothelial migration assay, sLR11 also markedly increased the migration of HL-60 cells. [Display omitted] [Display omitted] Conclusions:sLR11 is a novel chemotactic agent for HL-60 cells and induces strong enhancement of G-CSF-mediated migration and adhesion. LR11 may play a key role in G-CSF-mediated HSC mobilization by stimulating cell migration from BM into peripheral circulation. Disclosures:No relevant conflicts of interest to declare.