Increase In Transendothelial Migration Research Articles

Alpha‐2c Adrenergic Receptor Promotes the Malignant Phenotype of Colon Cancer Cells

Cancer remains one of the leading causes of death worldwide, with colorectal cancer (CRC) being the third most common type. Accumulating evidence shows that stress plays a pivotal role in the malignant phenotype of CRC, by promoting hallmarks of CRC including cellular proliferation, migration, invasion, and angiogenesis. During stressful situations, the sympathetic nervous system is activated, causing an elevation in levels of catecholamines, including epinephrine which elicits its effects mainly by activating adrenoceptors. Interestingly, however, the role of alpha2C adrenergic receptor (a2c‐AR) in CRC malignancy remains obscure. Here, we proposed that activation of a2c‐AR potentiates the malignant phenotype of human CRC cells. Treatment of human colon cancer cell lines, SW480 and HT‐29, with increasing concentrations of a2‐AR specific agonist (UK 14,304; 10, 30 or 100 nM) for different time points (24, 48, or 72 hours) did not cause a significant change in proliferation. However, using monolayer scratch assay, a significant increase in migration was observed after treatment with UK 14,304 (100 nM) for 8 and 12 hours in both cell lines. This was further supported by a transwell migration assay where UK 14,304 increased cell migration. Pretreatment with a specific a2c‐AR antagonist (JP 1302; 100 nM) abolished the UK 14,304‐induced cell migration. This increase in migratory capacity was concomitant with increased Rho activation evident by translocation of Rho from cytoplasm to the membrane as well as via a Rhotekin‐binding activation assay. Moreover, treatment with UK 14,304 evoked actin reorganization, an element known to be a prerequisite for cell migration. The induced Rho activation and actin cytoskeletal rearrangement could be mediated by increased oxidative stress since treatment with UK 14,304 increased ROS production. Importantly, ROS is known to induce migration of these cells. Furthermore, adhesion of colon cancer cells to TNF‐alpha‐activated HUVECs was promoted by UK 14,304. This UK14,304‐induced adhesion was followed by a dramatic increase in transendothelial migration of SW480 through HUVECs, clearly indicating that a2c‐AR activation may promote intra‐ or extravasation. Because breaking the endothelial cell barrier requires matrix digestion, we measured the levels of matrix metalloproteinases (MMPs). Importantly, stimulation of a2c‐ARs by UK14,304 increased the production of MMP2 and MMP9, and JP 1302 abolished this UK14,304‐induced production. Taken together, our data suggests that a2c‐AR potentiates the migratory and invasive capacities and hence the malignant phenotype of human colon cancer cells. Therefore, a2c‐AR may represent a novel target in the treatment of CRC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation of transendothelial migration of colon cancer cells by E-selectin-mediated activation of p38 and ERK MAP kinases

The invasive properties of cancer cells depend on their intrinsic motile potential and on their ability to breach the endothelial barrier. In the present work, we investigated the mechanisms by which adhesion of colon cancer cells to E-selectin expressed by endothelial cells regulates the barrier function of these cells and modulates transmigration of cancer cells. We found that the stimulation of E-selectin by activating antibodies or the adhesion of HT-29 cells results in an increase in the activity of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases. In turn, the activation of p38 and ERK enhances transendothelial permeability and migration of HT-29 cells. We also obtained evidence suggesting that p38-mediated increase in transendothelial migration of cancer cells depends on a myosin light chain phosphorylation-mediated formation of stress fibres. On the other hand, the activation of ERK by E-selectin modulates the opening of interendothelial spaces by initiating the activation of Src kinase activities and the dissociation of the VE-cadherin/beta-catenin complex. We conclude that activation of E-selectin by adhering cancer cells is an important process that regulates the extravasation of colon cancer cells by initiating p38- and ERK-dependent mechanisms that both contribute to regulate the integrity of the endothelial layer.

Exercise paradoxically modulates oxidized low density lipoproteininduced adhesion molecules expression and trans-endothelial migration of monocyte in men

Physical exercise can affect the risk of cardiovascular disease. Oxidized-low density lipoprotein (ox-LDL) promotes transendothelial migration (TEM) of monocyte, thereby accelerating the pathogenesis of atherosclerosis. This study investigated how exercise intensity affects monocyte/EC interactions under ox-LDL-mediated condition. Light- (LIE), moderate- (MIE) and high- (HIE) intensity exercise (i.e., 40%, 60%, and 80% VO2max, respectively) on a bicycle ergometer in 18 sedentary healthy men were performed on three separate occasions. Before and immediately after exercise, ox-LDL-promoted expressions of monocyte adhesion molecules and TEM of monocyte, as well as oxidation of LDL and amounts of soluble adhesion molecules in plasma were measured. Analytical results showed that (1) ox-LDL furthered monocyte L-selectin shedding and Mac-1 expression, and an attendant increase in TEM of monocyte, while treating the monocyte with Mac-1 antibody inhibited the ox-LDL-promoted TEM of monocyte; (2) under ox-LDL-treated condition, MIE increased monocyte Mac-1 and LFA-1 expressions, enhancing the TEM of monocyte, whereas HIE downregulated monocyte Mac-1 expression, suppressing the TEM of monocyte; (3) LIE decreased basal LFA-1 expression as well as basal and ox-LDL-promoted TEM of monocyte; and (4) MIE and HIE, but not LIE, elevated plasma ox-LDL level, while there were no significant changes in sL-selectin, sE-selectin, sICAM-1, and sVCAM-1 following these exercises. Therefore, we conclude that monocyte activation and subsequent TEM promoted by ox-LDL are changed by short-term exercise in an intensity-dependent manner. These findings provide a new insight into the may aid the development of suitable exercise intensity enable people to prevent early atherogenesis.

Modulation of heparan sulfate/chemokine interactions using RNA interference

Introduction The glycosaminoglycan heparan sulfate (HS) on the luminal surface of the vascular endothelium plays a vital role in promoting leucocyte migration during inflammation via specific interaction chemokines. HS is modified by sulfation and this appears to regulate chemokine binding. We previously observed that under pro‐inflammatory conditions [tumour necrosis factor α (TNF‐α) and interferon‐γ (IFN‐γ)], the endothelial cell line HMEC‐1 showed an increased sulfation HS. This promoted a significant increase binding of the chemokine RANTES and transendothelial migration of leucocytes. TNF‐α and IFN‐γ clearly modulated HS biosynthesis, in part by up‐regulating the NDST 1 and 2 genes responsible for N‐sulfation of the glycan, resulting in structural changes that favour HS/chemokine interactions (Carter et al. 2003). We have attempted to down‐regulate the expression of the gene NDST1 by RNA interference in order to investigate the structural and functional effects of reduced HS N‐sulfation.Materials and methods A double‐stranded interference RNA was designed and synthesized in vitro to target the sulfotransferase NDST‐1 and used to tranfect the HMEC‐1 cell line. A number of assays measured its effect on HS structure and function. Cell surface N‐sulfation was measured by flow cytometry using the antibody F58 10E4 (Seikagaku, Japan). Cell surface binding of the chemokine MCP‐1 was visualized by immunohistochemistry. The functional effect of MCP‐1 binding was measured using a trans‐endothelial migration assay.Results Surprisingly, the transfected cells appeared to show a small but marked increase in cell surface N‐sulfation. This was consistent with previous results transfecting a full‐length NDST‐1 antisense construct into the cell line, A549 (unpublished). The increase in N‐sulfation observed was supported functional date. Namely, transfected cells show an increased binding of the chemokine MCP‐1 and this promoted a statistically significant increase in transendothelial migration of leucocytes.Discussion The results observed were contrary to expectations. It would appear that the interference RNA molecule is in some way interfering in the regulation of the NDST‐1 expression leading to an increase not decrease in gene expression. This has yet to be verified but we are beginning real time expression studies to validate this. We have hypothesized two possible reasons for this observation; endothelial cells may be able to sense a reduction in NDST activity and up‐regulate gene expression. Or the presence of the interference RNA itself promotes NDST‐1 expression. The latter is supported by growing evidence that short double‐stranded RNAs are thought to up‐regulate the pro‐inflammatory STAT transcription factor family (Shuey et al. 2002).

Dopamine attenuates neutrophil–endothelial interaction and is a putative anti-inflammatory cytokine

Abstract Background Activated neutrophil (PMN) adherence to vascular endothelium comprises a key step for both transendothelial migration and initiation of potentially deleterious release of PMN products. Previous work has shown that the biogenic amine dopamine (DA) functions as an anti-inflammatory cytokine by inducing PMN apoptosis. The effect of DA on PMN transendothelial migration and adhesion receptor expression as well as on the endothelial molecules, E-selectin and intercellular adhesion molecule (ICAM) 1, was evaluated. Methods PMNs were isolated from healthy controls, stimulated with lipopolysaccharide (LPS) and tumour necrosis factor (TNF) α and treated with DA. CD11b and CD18 PMN adhesion receptor expression was assessed flow cytometrically. In a separate transwell experiment, the effect of the chemo-attractant peptide interleukin (IL) 8 on PMN migration was assessed. Human umbilical vein endothelial cells were stimulated with LPS/TNF-α, incubated with DA, and ICAM-1 and E-selectin endothelial molecule expression was assessed. Results There was a significant increase in transendothelial migration in stimulated PMNs as well as in CD11b/CD18, endothelial E-selectin and ICAM-1 expression compared with normal PMNs. DA attenuated these changes. The chemoattractant effect of IL-8 was also abolished. Conclusion These results show that DA attenuates the initial interaction between PMNs and endothelium, providing further evidence that it also functions as an anti-inflammatory cytokine.

Taurine Attenuates LPS-Induced Rolling and Adhesion in Rat Microcirculation

Background. Adhesion of polymorphonuclear leukocytes (PMN) to endothelial cells and subsequent transendothelial migration are an early key events in the inflammatory response and play an important part in the pathogenesis of septic shock, contributing to vascular and tissue injury. Taurine (2-aminoethanesulfonic acid) is a sulphur-containing β amino acid. It is a known antioxidant, possesses antimicrobial properties, and has previously been shown to be protective to the endothelium both in vivo and in vitro. The aim of this study was to determine if pretreatment with taurinewould attenuate the lipopolysaccharide (LPS)-induced increase in leukocyte-endothelial interactions and microvascular permeability during endotoxemia.Materials and methods. Male Sprague-Dawley rats (300–350 g) were randomized into three groups: (1) Control, (2) LPS, and (3) LPS + Taurine groups. Taurine was administered orally as a 4% solution. Endotoxemia was induced using Escherichia Coli endotoxin (Serotype 0.55 B5)—15 mg/kg via a slow intravenous infusion. Using mesenteric postcapillary venules (28–32-μm diameter) the number of adherent and migrated leukocytes and their rolling velocity were measured by intravital microscopy at baseline and subsequently at 10, 30, 60, and 90 min post administration of LPS.Results. Following administration of LPS there was a reduction in leukocyte rolling velocity at 30, 60 and 90 min. This was accompanied by a significant increase in the number of adherent leukocytes at 10, 30, 60 and 90 min. Transendothelial migration was significantly increased at 90 min. Taurine significantly attenuated the LPS-induced reduction in leukocyte rolling velocity at 10 and 30 min and the number of adherent leukocytes at all time points. Taurine also attenuated the LPS-induced increase in transendothelial migration at 90 min.Conclusions. These results demonstrate that taurine ameliorates endotoxin-induced leukocyte-endothelial cell interactions associated with sepsis, thereby suggesting that taurine may have a therapeutic role in the preventionof endothelial damage in sepsis.

Exposure to febrile temperature modifies endothelial cell response to tumor necrosis factor-alpha.

Fever is an important regulator of inflammation that modifies expression and bioactivity of cytokines, including tumor necrosis factor (TNF)-alpha. Pulmonary vascular endothelium is an important target of TNF-alpha during the systemic inflammatory response. In this study, we analyzed the effect of a febrile range temperature (39.5 degrees C) on TNF-alpha-stimulated changes in endothelial barrier function, capacity for neutrophil binding and transendothelial migration (TEM), and cytokine secretion in human pulmonary artery endothelial cells (EC). Permeability for [(14)C]BSA tracer was increased by treatment with TNF-alpha, and this effect was augmented by incubating EC at 39.5 degrees C. Treating EC with 2. 5 U/ml TNF-alpha stimulated an increase in subsequent neutrophil adherence and TEM. Incubating EC at 39.5 degrees C caused a 30% increase in TEM but did not modify the enhancement of neutrophil adherence or TEM by TNF-alpha treatment. Analysis of cytokine expression in EC cultures exposed to TNF-alpha at either 37 degrees or 39.5 degrees C revealed three patterns of temperature and TNF-alpha responsiveness. Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-8 were not detectable in untreated EC but were increased after TNF-alpha exposure, and this increase was enhanced at 39.5 degrees C. IL-6 expression was also increased with TNF-alpha exposure, but IL-6 expression was lower in 39.5 degrees C EC cultures. Transforming growth factor-beta(1) was constitutively expressed, and its expression was not influenced either by TNF-alpha or exposure to 39.5 degrees C. These data demonstrate that clinically relevant shifts in body temperature might cause important changes in the effects of proinflammatory cytokines on the endothelium.

Dopamine attenuates the chemoattractant effect of interleukin-8: a novel role in the systemic inflammatory response syndrome.

Activated neutrophil (PMN) adherence to vascular endothelium comprises a key step for both transendothelial migration and initiation of potentially deleterious release of PMN products. The biogenic amine, dopamine (DA), has been used for several decades in patients to maintain hemodynamic stability. The effect of dopamine on PMN transendothelial migration and adhesion receptor expression and on the endothelial molecules, E-selectin and ICAM-1, was evaluated. PMN were isolated from healthy controls, stimulated with lipopolysaccharide (LPS), and tumor necrosis factor-alpha (TNF-alpha) and treated with dopamine. CD 11b and CD 18 PMN adhesion receptor expression were assessed flow cytometrically. In a separate experiment, the chemoattractant peptide, IL-8, was placed in the lower chamber of transwells, and PMN migration was assessed. Human umbilical vein endothelial cells (HUVEC) were stimulated with LPS/TNF-alpha and incubated with dopamine. ICAM-1 and E-selectin endothelial molecule expression were assessed flow cytometrically. There was a significant increase in transendothelial migration in stimulated PMN compared with normal PMN (40 vs. 14%, P < 0.001). In addition, PMN CD11b/CD18 was significantly upregulated in stimulated PMN compared with normal PMN (252.4/352.4 vs. 76.7/139.4, P < 0.001) as were endothelial E-selectin/ICAM-1 expression compared with normal EC (8.1/9 vs. 3.9/3.8, P < 0.05). After treatment with dopamine, PMN transmigration was significantly decreased compared with stimulated PMN (8% vs. 40%, P < 0.001). Furthermore, dopamine also attenuated PMN CD11b/CD18 and the endothelial molecules E-selectin and ICAM-1 compared with stimulated PMN/EC that were not treated dopamine (174/240 vs. 252/352, P < 0.05 and 4/4.4 vs. 8.1/9, P < 0.05. respectively). The chemoattractant effect of IL-8 was also attenuated. These results identify for the first time that dopamine attenuates the initial interaction between PMN and the endothelium, and consequently, modulates PMN exudation. Thus, biogenic amines, including dopamine, may function as anti-inflammatory cytokines.

Characterization of the priming effect by pituitary canine growth hormone on canine polymorphonuclear neutrophil granulocyte function.

In this report, we demonstrate that canine growth hormone (cGH) is capable of priming canine polymorphonuclear neutrophil granulocytes (PMN) in a manner resembling that of human PMN. The cGH influences important functions that are involved in the process of recruitment of PMN, i.e., shape change, chemotaxis, CD11b/CD18 expression, adhesion, and subsequent transendothelial migration. Also, intracellular O(2)(-) production was evaluated. We investigated the priming effect by incubating PMN with purified pituitary cGH at various concentrations (10 to 800 microg/liter). The capacity for shape change was significantly (P < 0.05) enhanced, whereas the chemotactic response under agarose was significantly (P < 0.05) reduced. The chemotactic migration in Boyden chambers (10-microm-thick polycarbonate filter; lower surface count technique) was significantly (P < 0.05) enhanced, presumably due to cGH-induced hyperadhesiveness to the lower surface of the filters. The adhesion in albumin-coated microtiter plates and adherence to canine pulmonary fibroblasts were significantly (P < 0.05) increased, and the increased adhesion resulted in a significant (P < 0.01) increase in transendothelial migration using canine jugular vein endothelial cells. The increase in adhesion was associated with a significant increase in CD11b/CD18 expression. Furthermore, intracellular O(2)(-) production was significantly enhanced in response to both phorbol myristate acetate (P < 0.01) and opsonized zymosan (P < 0.05). In the absence of a PMN-stimulating agent, cGH did not influence the effector functions investigated except for an increased expression of CD11b/CD18.

CD3 directed bispecific antibodies induce increased lymphocyte-endothelial cell interactions in vitro.

Bispecific antibody (BsMAb) BIS-1 has been developed to redirect the cytolytic activity of cytotoxic T lymphocytes (CTL) to epithelial glycoprotein-2 (EGP-2) expressing tumour cells. Intravenous administration of BIS-1 F(ab′)2to carcinoma patients in a phase I/II clinical trial, caused immunomodulation as demonstrated by a rapid lymphopenia prior to a rise in plasma tumour necrosis factor-α and interferon-γ levels. Yet, no lymphocyte accumulation in the tumour tissue and no anti-tumour effect could be observed. These data suggest a BsMAb-induced lymphocyte adhesion to blood vessel walls and/or generalized redistribution of the lymphocytes into tissues. In this study, we describe the effects of BIS-1 F(ab′)2binding to peripheral blood mononuclear cells (PBMC) on their capacity to interact with resting endothelial cells in vitro. Resting and pre-activated PBMC exhibited a significant increase in adhesive interaction with endothelial cells when preincubated with BIS-1 F(ab′)2, followed by an increase in transendothelial migration (tem). Binding of BIS-1 F(ab′)2to PBMC affected the expression of a number of adhesion molecules involved in lymphocyte adhesion/migration. Furthermore, PBMC preincubated with BIS-1 F(ab′)2induced the expression of endothelial cell adhesion molecules E-selectin, VCAM-1 and ICAM-1 during adhesion/tem. These phenomena were related to the CD3 recognizing antibody fragment of the BsMAb and dependent on lymphocyte–endothelial cell contact. Possibly, in patients, the BIS-1 F(ab′)2infusion induced lymphopenia is a result of generalized activation of endothelial cells, leading to the formation of a temporary sink for lymphocytes. This process may distract the lymphocytes from homing to the tumour cells, and hence prevent the occurrence of BIS-1 F(ab′)2– CTL-mediated tumour cell lysis. © 2000 Cancer Research Campaign

Endothelial cell adhesion molecule and PMNL response to inflammatory stimuli and AGE-modified fibronectin

Atherosclerotic vascular disease is the leading cause of death in patients with diabetes mellitus and end-stage renal disease. Advanced glycation end products (AGEs) are strongly suggested to be involved in the pathogenesis of atherosclerosis in these patients who also frequently experience infectious complications. We hypothesized that the interaction of AGEs and inflammatory mediators contributes to the up-regulation of endothelial cell activation. We investigated the effect of advanced glycated fibronectin in the presence or absence of inflammatory stimuli on the endothelial cell surface and mRNA expression of cell adhesion molecules. Furthermore, the influence of advanced glycated fibronectin on the transendothelial migration pattern of polymorphonuclear cells was analyzed. Exposure to advanced glycated fibronectin together with inflammatory stimuli such as interleukin (IL)-1alpha, tumor necrosis factor-alpha (TNF-alpha) or lipopolysaccharide (LPS) led to a significant increase in the surface expression of the cell adhesion molecules E-selectin, ICAM-1, VCAM-1 and PECAM-1 on endothelial cells. Soluble AGEs in combination with advanced glycated fibronectin significantly enhanced the endothelial cell surface expression of ICAM-1, VCAM-1 and PECAM-1, whereas this was not the case for E-selectin. At the transcriptional level short-time exposure of endothelial cells to advanced glycated fibronectin and inflammatory mediators resulted in an increased expression of E-selectin, ICAM-1 and VCAM-1 mRNA levels, whereas PECAM-1 repeatedly showed a significant decrease of gene transcript levels. An increase of mRNA levels was also observed for E-selectin, ICAM-1, VCAM-1 and PECAM-1 following incubation with a combination of advanced glycated fibronectin and soluble advanced glycation end-products. Furthermore, polymorphonuclear cells responded with a sevenfold increase in transendothelial migration following exposure of endothelial cells to advanced glycated fibronectin and inflammatory mediators. These results suggest that the combination of matrix glycation and inflammation up-regulates the activation of the endothelial cell adhesion cascade, a mechanism that might contribute to the increased burden of atherosclerotic morbidity and mortality in patients suffering from diabetes mellitus or chronic renal failure.