Process Of Transendothelial Migration Research Articles

Transendothelial migration of the Lyme disease spirochete involves spirochete internalization as an intermediate step through a transcellular pathway that involves Cdc42 and Rac1.

Despite its importance in pathogenesis, the hematogenous dissemination pathway of Borrelia burgdorferi is still largely uncharacterized. To probe the molecular details of transendothelial migration more easily, we studied this process using cultured primary or telomerase-immortalized human microvascular endothelial cells in a medium that maintains both the human cells and the spirochetes. In B. burgdorferi-infected monolayers, we observed ~55% of wild-type spirochetes crossing the monolayer. Microscopic characterization revealed entrance points across the cellular surface rather than at cellular junctions, supporting a transcellular route. In support of this pathway, locking the endothelial junctions using a vascular endothelial protein tyrosine phosphatase (VE-PTP) inhibitor did not reduce transendothelial migration. We also used inhibitors to block the most common endocytic pathways to elucidate effectors that might be involved in B. burgdorferi uptake and/or transmigration. Directly inhibiting Cdc42 reduced spirochete transmigration by impeding internalization. However, blocking Rac1 alone dramatically reduced transmigration by ~84% and resulted in a concomitant doubling in spirochete accumulation in the cell. Our combined results support that B. burgdorferi internalization is an intermediate step in the transendothelial migration process, which requires both Cdc42 and Rac1; Cdc42 is needed for spirochete internalization, while Rac1 is required for cellular egress. These are the first two host proteins implicated in B. burgdorferi transmigration across endothelial cells.IMPORTANCELyme borreliosis is caused by Borrelia burgdorferi and related bacteria. It is the most common tick-transmitted illness in the Northern Hemisphere. The ability of this pathogen to spread to a wide variety of locations results in a diverse set of clinical manifestations, yet little is known regarding vascular escape of the spirochete, an important pathway for dissemination. Our current work has studied the traversal of B. burgdorferi across a monolayer of microvascular endothelial cells grown using a new culture system. We show that this occurs by passage of the spirochetes directly through cells rather than at cellular junctions and that internalization of B. burgdorferi is an intermediate step in transmigration. We also identify the first two host proteins, Cdc42 and Rac1, that are used by the spirochetes to promote traversal of the cellular monolayer. Our new experimental system also provides a new avenue for further studies of this important process.

Effect of Enamel Matrix Derivatives on Osteoclast Formation from PBMC of Periodontitis Patients and Healthy Individuals after Interaction with Activated Endothelial Cells.

Background and objectives: Enamel matrix derivative (EMD) is produced from developing porcine tooth buds and represents a complex of low-molecular-weight hydrophobic enamel proteins. EMD is widely applied in periodontal regeneration. Osteoclasts are multinuclear cells, which are responsible for bone resorption. The precursors of osteoclasts, hematopoietic cells, undergo in vivo the process of transendothelial migration before differentiation. EMD is known to affect the process of osteoclastogenesis, but its effect on human osteoclasts precursors after the interaction with activated endothelium was never studied. Materials and Methods: Human umbilical vein endothelial cells (HUVECs)s were seeded in transwell inserts with a pore size of 8 µm and pre-activated by TNF-α and IL-1β for 18 h. Peripheral blood mononuclear cells (PBMCs), freshly isolated from 16 periodontitis patients and 16 healthy individuals, were added to pre-activated HUVECs. Adherent, non-adherent and transmigrated cells were collected and differentiated to osteoclasts by the standard protocol in the presence or absence of EMD. The number of osteoclasts was determined by tartrate-resistant acid phosphatase staining. Results: PBMCs isolated from periodontitis patients have formed a significantly higher osteoclast number compared to PBMCs isolated from healthy individuals (p < 0.05). EMD induced concentration-dependent inhibition of osteoclast formation from PBMCs. This was true for the different PBMC fractions isolated from both healthy individuals and periodontitis patients. Conclusions: Our data show that EMD inhibits the formation and activity of osteoclasts differentiated from the progenitor cells after the interaction with activated endothelium. This might be associated with bone resorption inhibition and supporting bone regeneration in the frame of periodontal therapy.

Functionally distinct IFN-γ+ IL-17A+ Th cells in experimental autoimmune uveitis: T-cell heterogeneity, migration, and steroid response.

Immunopathogenic roles for both Th1 (CD4+ IFN-γ+ ) and Th17 (CD4+ IL-17A+ ) cells have been demonstrated in experimental autoimmune uveitis (EAU). However, the role for Th17/Th1 (CD4+ T cells co-expressing IFN-γ and IL-17A) cells in EAU is not yet understood. Using interphotoreceptor retinoid-binding protein peptide-induced EAU in mice, we found increased levels of Th17/Th1 cells in EAU retinae (mean 9.6±4.2%) and draining LNs (mean 8.4±3.9%; p=0.01) relative to controls. Topical dexamethasone treatment effectively reduced EAU severity and decreased retinal Th1 cells (p=0.01), but had no impact on retinal Th17/Th1 or Th17 cells compared to saline controls. Using in vitro migration assays with mouse CNS endothelium, we demonstrated that Th17/Th1 cells were significantly increased within the migrated population relative to controls (mean 15.6±9.5% vs. 1.9±1.5%; p=0.01). Chemokine receptor profiles of Th17/Th1 cells (CXCR3 and CCR6) did not change throughout the transendothelial migration process and were unaffected by dexamethasone treatment. These findings support a role for Th17/Th1 cells in EAU and their resistance to steroid inhibition suggests the importance of targeting both Th17 and Th17/Th1 cells for improving therapy.

Peculiarities of the expression of the vascular adhesion molecule in acute IA phase pancreatitis

Purpose : to estimate the expression of a molecule of vascular adhesion of PECAM-1 (CD31) at patients with acute pancreatitis of varying severity in ΙА phase diseasedevelopment. Materials and methods . Work has clinical character and is based on the prospective analysis of observation and treatment of 135 patients with acute pancreatitis of various degrees of severity. Patients were divided into three groups, depending on severity of a disease: 1 group — 45 patients with slight acute pancreatitis, the 2 nd group — 45 patients with medium-weight acute pancreatitis, the 3 rd group — 45 patients with heavy acute pancreatitis. Blood sampling for a research was carried out at receipt prior to therapy. Phenotyping of lymphocytes was carried out by method of an indirect immunofl uorescence by means of mouse monoclonal antibodies to the molecules CD31 receptors of lymphocytes. Results . The obtained data demonstrate that the percent of CD31-expressing of lymphocytes depends on weight of acute pancreatitis. At initially severe form of a disease, there was strengthening of process of transendothelial migration of lymphocytes in the center of forming of a necrosis in pancreas tissue. In our research signifi cantly (p &lt;0.001, p1 &lt;0.001, p2 &lt;0.001) the percent of the CD31-expressing lymphocytes to 13.21 increased [12.50; 5.76]. Conclusion . The obtained data demonstrate that there is an interrelation between PECAM1 expression as a marker of intercellular interactions and severity of acute pancreatitis.

3307Impaired reverse transendothelial migration capacity governs the enhanced abluminal accumulation of diabetic CD14+ monocytes

Abstract Background Chronic inflammation drives atherosclerosis. Monocyte recruitment and accumulation in the tissues contributes to chronic inflammation and is an important aspect of the pathobiology of atherosclerosis. Diabetes mellitus (DM) is a leading cardiovascular predisposing to the development of atherosclerosis. Monocytes represent the largest cell-population in the atherosclerotic plaque. However, the mechanisms that drive the accumulation of monocytes in the plaques are not clearly understood. Purpose The aim of this study is to understand the hyperglycemia/diabetes-induced changes in the transendothelial and reverse transendothelial migration processes of monocytes under physiological flow conditions and to characterize the underlying mechanisms. Methods Monocytes isolated from healthy donors by negative selection were conditioned in hyperglycemic or normoglycemic conditions for 48 hours. These monocytes or monocytes from type 2 diabetes mellitus (T2-DM) patients or from non-diabetic controls were allowed to interact with TNFα -activated HUVEC-monolayer. Experiments were performed under flow conditions found in post-capillary venules. Analysis of migration was performed using Fiji J software. Expression of molecules involved in transmigration and reverse migration was measured using FACS. Results Monocytes from T2-DM patients and hyperglycemia-conditioned cells revealed a significantly reduced transendothelial migration capacity. Even though these monocytes adhered to the HUVEC monolayer, only few of them transmigrated. Moreover, the transmigrated cells from diabetic patients and hyperglycemia-conditioned cells accumulated in the ablumen after transendothelial migration. Time in the ablumen was prolonged and there was a significant reduction in the reverse transendothelial migration. Expression levels of chemokine receptors, which had been shown to be essential for transendothelial migration process, were reduced in diabetic monocytes. Conclusion Our results revealed for the first time that the enhanced diabetic monocyte accumulation in the albumen is not secondary to the elevated transmigration through the endothelium. Instead, the accumulation of monocytes in the vessel wall is due to the direct consequence of dysfunctional reverse transendothelial migration. These novel findings could explain the underlying reasons leading to the elevated inflammatory monocyte accumulation, chronic inflammation and subsequent atherosclerosis development in T2-DM patients. Our results highlight the importance of restoring the reverse transendothelial migration capacity of monocytes to reduce monocyte accumulation and atherogenesis in the diabetic environment.

Endothelial RhoB and RhoC are dispensable for leukocyte diapedesis and for maintaining vascular integrity during diapedesis

ABSTRACTActive remodeling of the actin cytoskeleton in endothelial cells is necessary for allowing leukocytes to cross the barrier during the process of transendothelial migration (TEM). Involvement of RhoGTPases to regulate actin organization is inevitable, and we recently reported on the local function of RhoA in limiting vascular leakage during leukocyte TEM. As a follow-up we investigated here the possible involvement of two other closely-related GTPases; RhoB and RhoC, in regulating leukocyte TEM and vascular barrier maintenance. Physiological flow experiments showed no substantial involvement of either endothelial RhoB or RhoC in neutrophil adhesion and transmigration efficiency. Besides neutrophil TEM, we did not observe a role for endothelial RhoB or RhoC in limiting vascular leakage in both inflammatory conditions and during TEM. In conclusion, endothelial RhoB and RhoC are both dispensable for regulating leukocyte diapedesis and for maintaining vascular barrier function under inflammatory conditions and during leukocyte diapedesis.

Potential role of CXCL9 induced by endothelial cells/CD133+ liver cancer cells co-culture system in tumor transendothelial migration.

Transendothelial migration is a pivotal step before the dissemination of tumor cells into the blood circulation. Related researches about the crosstalk between tumor cells and endothelial cells could contribute to understanding the mechanism of transendothelial migration. Cumulative studies showed that CD133 was an important marker for cancer stem cells. In our research, a co-culture system was developed to study the interaction between CD133+ liver cancer cells and human umbilical vein endothelial cells. The results showed that the direct co-cultured supernatants promoted the migration and invasion of CD133+ liver cancer cells. It was further investigated that the expression level of chemokine CXCL9 was significantly elevated in the culture supernatants of direct co-culture system by activating the NF-kB, rather than in the indirect co-culture system or mono-culture system. High expression of CXCL9 in the direct co-cultured supernatants played a significant role in enhancing the migration and invasion of CD133+ liver cancer cells. Collectively, these findings suggest that chemokine CXCL9 may function as a potential target during the process of transendothelial migration.

Adapting coculture in vitro models of the blood–brain barrier for use in cancer research: maintaining an appropriate endothelial monolayer for the assessment of transendothelial migration

Although brain metastases are the most common brain tumors in adults, there are few treatment options in this setting. To colonize the brain, circulating tumor cells must cross the blood–brain barrier (BBB), which is situated within specialized, restrictive microvascular endothelium. Understanding how cancer cells manage to transmigrate through the BBB might enable this process to be prevented. In vitro models are dedicated tools for characterizing the cellular and molecular mechanisms that underlie transendothelial migration process, as long as they accurately mimic the brain endothelium's in vivo characteristics. The objective of the present study was to adapt an existing in vitro model of the human BBB for use in studying cancer cell transmigration. The model is based on the coculture of endothelial cells (ECs, derived from cord blood hematopoietic stem cells) and brain pericytes. To allow the migration of cancer cells into the lower compartment, our model had to be transposed onto inserts with a larger pore size. However, we encountered a problem when culturing ECs on large (3-μm)-pore inserts: the cells crossed the membrane and formed a non-physiological second layer on the lower face of the insert. Using 3-μm-pore inserts (in a 12-well plate format), we report here on a method that enables the maintenance of a single monolayer of ECs on the insert's upper face only. Under these chosen conditions, the ECs exhibited typical BBB properties found in the original model (including restricted paracellular permeability and the expression of continuous tight junctions). This modified in vitro model of the human BBB enabled us to investigate the migratory potential of the MDA-MB-231 cell line (derived from highly metastatic human breast cancer cells). Last, the results obtained were compared with the rate of transmigration through endothelia with no BBB features.

Myosin-IIA regulates leukemia engraftment and brain infiltration in a mouse model of acute lymphoblastic leukemia.

Leukemia dissemination (the spread of leukemia cells from the bone marrow) and relapse are associated with poor prognosis. Often, relapse occurs in peripheral organs, such as the CNS, which acts as a sanctuary site for leukemia cells to escape anti-cancer treatments. Similar to normal leukocyte migration, leukemia dissemination entails migration of cells from the blood circulation into tissues by extravasation. To extravasate, leukemia cells cross through vascular endothelial walls via a process called transendothelial migration, which requires cytoskeletal remodeling. However, the specific molecular players in leukemia extravasation are not fully known. We examined the role of myosin-IIA a cytoskeletal class II myosin motor protein, in leukemia progression and dissemination into the CNS by use of a mouse model of Bcr-Abl-driven B cell acute lymphoblastic leukemia. Small hairpin RNA-mediated depletion of myosin-IIA did not affect apoptosis or the growth rate of B cell acute lymphoblastic leukemia cells. However, in an in vivo leukemia transfer model, myosin-IIA depletion slowed leukemia progression and prolonged survival, in part, by reducing the ability of B cell acute lymphoblastic leukemia cells to engraft efficiently. Finally, myosin-IIA inhibition, either by small hairpin RNA depletion or chemical inhibition by blebbistatin, drastically reduced CNS infiltration of leukemia cells. The effects on leukemia cell entry into tissues were mostly a result of the requirement for myosin-IIA to enable leukemia cells to complete the transendothelial migration process during extravasation. Overall, our data implicate myosin-IIA as a key mediator of leukemia cell migration, making it a promising target to inhibit leukemia dissemination in vivo and potentially reduce leukemia relapses.

Recapitulation of in vivo-like neutrophil transendothelial migration using a microfluidic platform.

Neutrophil transendothelial migration (TEM) is an essential physiological process that regulates the recruitment of neutrophils in response to inflammatory signals. Herein, a versatile hydrogel scaffold is embedded in a microfluidic platform that supports an endothelial cell layer cultured in the vertical direction and highly stable chemical gradients; this construct is employed to mimic the in vivo neutrophil TEM process. We found that the number of neutrophils migrating across the endothelial cell layer is dependent on the presented chemoattractant concentration and the spatial profile of the chemical gradient. Endothelial cells play a critical role in neutrophil TEM by promoting neutrophil morphological changes as well as expressing surface receptor molecules that are indispensable for inducing neutrophil attachment and migration. Furthermore, the microfluidic device also supports competing chemoattractant gradients to facilitate neutrophil TEM studies in complex microenvironments that more accurately model the in vivo system than simplified microenvironments without the complexity of chemical gradients. This work demonstrates that combinations of any two different chemoattractants induce more significant neutrophil migration than a single chemoattractant in the same total amount, indicating synergistic effects between distinct chemoattractants. The in vitro reconstitution of neutrophil TEM successfully translates planar neutrophil movement into in vivo-like neutrophil recruitment and accelerates understanding of cellular interactions between neutrophils and endothelial cells within the complicated physiological milieu.

Symposium 8: Cardiovascular nutrigenomics: Perspectives for ageing, healthspan and beyond S8-1 - MicroRNAs as novel nutrigenomic targets for cardiovascular health

Consumption of flavanol-rich foods is associated with a reduced risk of cardiovascular diseases, which was linked to improvements in endothelial function. The specific flavanols involved in these beneficial effects and underlying molecular mechanisms is still largely unknown. We have shown that exposure of TNFα-activated endothelial cells to flavanol metabolites (4'-O-methyl(-)-epicatechin, 4'-O-methyl(-)-epicatechin-7-β-D-glucuronide and (-)-epicatechin-4'-sulfate) at physiologically-relevant concentrations decreased the adhesion of monocytes to endothelial monolayers. Nutrigenomic analysis showed that these metabolites modulate expression of genes involved in the regulation of cell adhesion/junctions, focal adhesion or cytoskeleton remodeling, and this by affecting phosphorylation levels of p65 and p38 of NF-κB and MAPK cell-signaling pathways respectively. Together with cell signaling pathways, microRNAs (short, endogenous, noncoding, single-stranded RNAs) represent another class of molecular post-transcriptional regulators of gene expression. Our nutrigenomic studies have shown that exposure of endothelial cells to the same metabolites can also modulate the expression of miRNAs. Among differentially-expressed miRNAs are those involved in the regulation of inflammation or cell adhesion, such as miR-221 and miR-181. Bioinformatic analysis shows that the potential target genes these miRNAs are also involved in regulation of cell adhesion, cytoskeleton, focal adhesion, transendothelial migration. These miRNAs could exert post-transcriptional regulation by inhibiting protein synthesis, as BIRC2, or by inducing mRNA degradation, as WASP1.These data suggest that miRNAs are important molecular targets of flavanol metabolites involved in the regulation of expression of genes controlling adhesion and transendothelial migration processes. This original result contributes to increase the knowledge about the mechanisms underlying the protective effect of flavanols on vascular endothelium.

Treatment of endothelial cell with flavonoids modulates transendothelial leukocyte migration.

This study aimed to investigate the influence of the flavonoid oxerutin (Venoruton®, Novartis, Basel, Switzerland) on endothelial cell apoptosis and transendothelial migration of peripheral blood mononuclear cells and to elucidate the potential mechanisms affecting these processes. Human endothelial cells were treated with Venoruton to assess the potential effect on apoptosis and on the transendothelial migration process. Endothelial nitric oxide synthase and inducible nitric oxide synthase expression in endothelial cell after Venoruton treatment as well as reactive oxygen species levels were analyzed. Low-dose Venoruton shows a protective effect on endothelial cells and inhibits transendothelial migration of peripheral blood mononuclear cells through an endothelial monolayer, but high-dose Venoruton inversely elevated transendothelial migration of peripheral blood mononuclear cells. Meanwhile, a dose-dependent action of Venoruton on endothelial cell apoptosis could be observed. Endothelial nitric oxide synthase and inducible nitric oxide synthase expression were gradually increased in endothelial cells with increasing Venoruton dosage. In addition, reactive oxygen species were significantly reduced by 0.1 mM and 0.5 mM Venoruton and elevated after high dose treatment. These data suggest that the increased transendothelial migration of peripheral blood mononuclear cells is related to the excessive activation of the nitric oxide-axis and subsequent relaxation of the endothelial cells.

Trisomy 12 CLL Cells Have High Surface Expression Of Integrins Involved In Lymphocyte Trafficking But This Does Not Translate Into Improved LFA-1-Mediated Motility

Chronic lymphocytic leukemia (CLL) is a disease of considerable clinical and genetic heterogeneity. Trisomy of chromosome 12 is the third-most common cytogenetic abnormality and has several distinguishing features including abnormal morphology, high prevalence of NOTCH1 mutations, high expression of CD11a and no additional clinical benefit from the addition of rituximab to fludarabine and cyclophosphamide. Previous studies have also reported a higher prevalence of trisomy 12 in patients with small lymphocytic lymphoma (SLL). The heterodimeric integrins CD18/CD11a (LFA-1), CD18/CD11b (Mac-1) and CD49d/CD29 (VLA-4) are cell surface transmembrane proteins involved in the inducible adhesion of leukocytes to vascular walls during the process of transendothelial migration from the bloodstream into the tissues. This process is particularly important in CLL as it allows the malignant cells to enter lymphoid organs where they receive growth and survival signals and are protected from chemotherapy.Multi-color flow cytometry was used to assess the cell surface expression of a panel of proteins involved in cell adhesion, motility and transendothelial migration. Compared to control B cells from the peripheral blood of age-matched healthy donors (n=25), CLL cells from peripheral blood of untreated CLL patients (n=101) had significantly reduced expression of CD11a, CD11b, CD18, CD29 and CD49d (p<0.0001). However, uniquely among the main cytogenetic categories, CLL cells from patients with trisomy 12 (n=12) had relatively preserved expression of these integrins, with levels comparable to healthy B cells. Notably, we confirm previous studies demonstrating the prognostic significance of integrin expression in CLL with high levels of CD11a and CD49d being associated with reduced time to first treatment.To examine the functional significance of the altered integrin expression on CLL cells, time-lapse microscopy was used to investigate the behaviour of tumor cells from CLL patients with or without trisomy 12, as well as B cells from healthy donors. Plates were coated with intercellular adhesion molecule (ICAM)-1 (LFA-1 ligand), and the cells stimulated with the chemokine CXCL12 (SDF-1). To measure cell motility, images were acquired using a Nikon Biostation IM microscope using a 20x objective lens at 30 second intervals for 40 minutes. A minimum of 50 individual cells per patient were tracked and their average velocity calculated using NIS-Element AR software. Although the average velocity of healthy B cells was significantly greater than CLL cells from all cytogenetic groups (mean 0.06119 µm/s, n=3 versus 0.03081 µm/s, n=8, p<0.0001), surprisingly, there was no significant difference in the average velocity of trisomy 12 and non-trisomy 12 CLL cells despite the differences in integrin expression (0.02980 µm/s, n=4 versus 0.03182 µm/s, n=4, p=0.3406). Since we have previously observed differences in rho-GTPases in CLL cells, we examined expression of the integrin-associated protein RAP1B as this is functionally associated with LFA-1, Mac-1 and VLA-4 and the gene is located on chromosome 12. We found no significant increase in the levels of RAP1B mRNA in trisomy 12 CLL cells compared to both healthy B cells and non-trisomy 12 CLL cells, although trisomy 12 CLL cells had higher RAP1B:RAP1A ratio. CLL cells with trisomy 12, but not other cytogenetic subgroups, further increased integrin expression following BCR-crosslinking and on-going studies are examining the impact of this within the tumor microenvironment.In conclusion, CLL cells from patients with trisomy 12 have a unique phenotype with relatively preserved expression of CD11a, CD11b, CD18, CD29 and CD49d. However, this increased integrin expression compared to non-trisomy 12 CLL cells does not result in improved LFA-1-mediated motility on ICAM-1. The motility defect of CLL cells may thus be indicative of a more general cytoskeletal defect that is independent of cell surface integrin expression. We continue to study mechanisms whereby altered expression of integrins on trisomy 12 CLL cells is associated with increased resistance to anti-CD20 mAb therapy, or whether this is instead mediated by differences in the tumor microenvironment. Disclosures:Gribben:Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria. Riches:Celgene: Research Funding.

Proteolysis during Tumor Cell Extravasation In Vitro: Metalloproteinase Involvement across Tumor Cell Types

To test if proteolysis is involved in tumor cell extravasation, we developed an in vitro model where tumor cells cross an endothelial monolayer cultured on a basement membrane. Using this model we classified the ability of the cells to transmigrate through the endothelial cell barrier onto the underlying matrix, and scored this invasion according to the stage of passage through the endothelium. Metalloproteinase inhibitors reduced tumor cell extravasation by at least 35%. Visualization of protease and cell adhesion molecules by confocal microscopy demonstrated the cell surface localization of MMP-2, MMP-9, MT1-MMP, furin, CD44 and αvβ3, during the process of transendothelial migration. By the addition of inhibitors and bio-modulators we assessed the functional requirement of the aforementioned molecules for efficient migration. Proteolytic digestion occurred at the cell-matrix interface and was most evident during the migratory stage. All of the inhibitors and biomodulators affected the transition of the tumor cells into the migratory stage, highlighting the most prevalent use of proteolysis at this particular step of tumor cell extravasation. These data suggest that a proteolytic interface operates at the tumor cell surface within the tumor-endothelial cell microenvironment.

Abstract A11: Redistribution of endothelial junction protein PECAM-1 and MMP activity are important for transmigration of breast tumor cells across lung endothelial monolayer in-vitro

Abstract The ability of tumor cells to escape the circulation is essential to the establishment of secondary malignancy. The occurrence of metastases in a particular secondary site is influenced by differences in the barriers present at each such site, as well as the unique organ-specific microenvironments. One of the preferred metastatic sites for breast cancer is lung and our study aims to identify in vitro the interactions between breast tumor cells and lung endothelium that may be important in the metastatic process. In this study, we used in vitro tumor binding assays and the transwell assay system to examine the adhesive interactions of breast tumor cells (MDA-MB-231, MCF-7) and subsequent transmigration across monolayers of endothelial cells. In addition, we compared breast cancer cell interactions with endothelial cells isolated from different organ sites, namely human lung (FLEC), saphenous vein (SVEC), and umbilical cord vein (HUVEC). To determine the mechanisms facilitating transmigration, we examined, using immunofluorescent staining, the distribution of the endothelial junction protein, PECAM-1, on tumor cell-bound endothelial monolayers. The role of matrix metalloproteinases (MMPs) in the transendothelial migration process was examined via various MMP(s) inhibitors. FLEC were able to bind both the highly invasive MDA-MB-231 and the non-metastasising MCF-7 cells with similar efficiency. However, more MDA-MB-231 transmigrated the FLEC monolayer as compared to MCF-7. Bound MDA-MB-231 cells were more effective at disrupting FLEC junction integrity, resulting in the formation of ‘gaps’ at cellular junctions with loss of PECAM-1 expression. Treatment with broad spectrum MMPs inhibitor (GM6001) and specific MMP2, 3, 8 inhibitors (OA-HY, MMP3 inhibitor II, MMP8 inhibitor I respectively) resulted in a reduction in MDA-MB-231 transmigration across the FLEC monolayer. A yet unidentified MMP(s) secreted by MDA-MB-231 and FLEC-derived MMP2 contributed to the observed ‘gap’ formation. In comparison, MDA-MB-231 transmigration across HUVEC and SVEC monolayers were significantly less efficient compared to FLEC monolayer. The resistance of HUVEC and SVEC to MDA-MB-231 transmigration correlated to significantly less disruption of junction integrity and gap formation by bound MDA-MB-231. Our data suggest that disruption of endothelial junction integrity, due to redistribution of PECAM-1 and MMP activity, contributes to breast tumor cell transmigration across lung endothelial monolayer. The decreased interaction of breast tumor cells with HUVEC and SVEC suggests that breast tumor cells preferentially interact with lung endothelial cells, thus resulting in the lung being a common site of metastasis in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A11.

The presence of alpha-catenin in the VE-cadherin complex is required for efficient transendothelial migration of leukocytes

The majority of the leukocytes cross the endothelial lining of the vessels through cell-cell junctions. The junctional protein Vascular Endothelial (VE)-cadherin is transiently re-distributed from sites of cell-cell contacts during passage of leukocytes. VE-cadherin is part of a protein complex comprising p120-catenin and beta-catenin as intracellular partners. Beta-catenin connects VE-cadherin to alpha-catenin. This VE-cadherin-catenin complex is believed to dynamically control endothelial cell-cell junctions and to regulate the passage of leukocytes, although not much is known about the role of alpha- and beta-catenin during the process of transendothelial migration (TEM). In order to study the importance of the interaction between alpha- and beta-catenin in TEM, we used a cell-permeable version of the peptide encoding the binding site of alpha-catenin for beta-catenin (S27D). The data show that S27D interferes with the interaction between alpha- and beta-catenin and induces a reversible decrease in electrical resistance of the endothelial monolayer. In addition, S27D co-localized with beta-catenin at cell-cell junctions. Surprisingly, transmigration of neutrophils across endothelial monolayers was blocked in the presence of S27D. In conclusion, our results show for the first time that the association of alpha-catenin with the cadherin-catenin complex is required for efficient leukocyte TEM.

Neutrophil Recruitment under Shear Flow: It's All about Endothelial Cell Rings and Gaps

Leukocyte recruitment to tissues and organs is an essential component of host defense. The molecular mechanisms controlling this process are complex and remain under active investigation. The combination of biochemical techniques and live cell imaging using in vivo and in vitro flow-model approaches have shed light on several aspects of neutrophil transmigration through the vascular endothelial lining of blood vessels. Here, we focus on the role of adhesion molecule signaling in endothelial cells and their downstream targets during the process of transendothelial migration at cell-cell borders (paracellular transmigration). An emerging model involves the leukocyte beta2 integrin engagement of endothelial cell ICAM-1, which triggers integrin-ICAM-1 clustering (rings) and stabilizes leukocyte adhesion at cell-cell junctions. This step recruits nonreceptor tyrosine kinases that phosphorylate key tyrosine residues in the cytoplasmic tail of VE-cadherin, which destabilizes its linkage to catenins and the actin cytoskeleton, triggering the transient opening of VE-cadherin homodimers to form a gap in the cell junction, through which the neutrophil transmigrates. Interestingly, the signaling events that lead to neutrophil transmigration occur independently of shear flow in vitro.

Expression profiling reveals genes associated with transendothelial migration of tumor cells: A functional role for αvβ3 integrin

Transendothelial migration is a key step in the extravasation of tumor cells during metastasis formation. Here, we have classified 45 human tumor cell lines derived from various tissues according to their capacity for transendothelial migration in vitro. We could distinguish cell lines showing strong transmigration (TEM+ cell lines) from others that did not transmigrate (TEM- cell lines). By DNA microarray analysis we could cluster TEM+ and TEM- cell lines according to their gene expression pattern and identify genes differentially expressed between the 2 groups. Among these we found the integrin beta3 subunit to be highly expressed in TEM+ cell lines as compared to TEM- cell lines. Cell surface localization of alphavbeta3 integrin receptors was exclusively found in TEM+ cell lines. Transendothelial migration of TEM+ cells but not their adhesion to the endothelial cells, or invasion into collagen gels could be blocked with an antibody against alphavbeta3 integrin and by RNAi mediated knock-down of the integrin beta3 subunit. These data establishes alphavbeta3 integrin as one key component of the transendothelial migration process of tumor cells, and as a potential target for anti-metastatic therapy. Our gene expression analysis of a defined collection of tumor cell lines can be used as a starting point to identify further genes functionally involved in transendothelial migration.

The Extramedullar Dissemination of Acute Myeloid Leukemic Cells Depends on CD31 (PECAM-1) and CD38 Coexpression Level.

The number of peripheral white blood cells (WBC) shows a wide range in acute myeloblastic leukemia (AML) patients. A high WBC count constitutes an adverse prognosis factor when at presentation and (myelo)monocytic subtypes were more frequently associated with extramedullar tumor sites. Marrow endothelial cells express CD31 and CD31/CD31 interaction is known to promote the migration in physiological mechanisms, as for example transendothelial migration of neutrophiles and monocytes in diapedesis. CD31 is a specific receptor of CD38 which is associated with numerous molecules on the surface of blood cells. Moreover, CD38 could interact with hyaluronate, a component of the extracellular matrix, 2 hyaluronate-binding sites having been reported in its extracellular domain. These elements lead us to postulate that CD31 and CD38 are colocalized on AML cells and that an excess of CD31 promotes the egress of the cells from the marrow compartment, while an excess of CD38 favors their anchorage to marrow microenvironment.The CD38/CD31 colocalization was demonstrated using FRET and cocapping strategies. FRET experiments were performed with a 488 nm laser flow cytometer, Cy3-conjugated anti-CD38 mAb and FITC-conjugated mAb against CD31. For cocapping experiments, we induced capping of CD31 with anti-CD31 mAb at 37°C; the cells were then fixed and labeled with anti-CD38 mAb. Transendothelial migration has been studied using anti-CD31 mAb and anti-CD38 blocking mAbs in Transwell® experiments performed with TrHBMEC cell line. To study the CD38/hyaluronate interaction, cells were treated or not with all-trans retinoic acid to induce CD38 overexpression just before plating the cells onto hyaluronate-coated dish. The in vivo influence of CD31 and CD38 coexpression level was evaluated by studying the phenotype of marrow leukemic cells (S/N ratio of the MFI) and the WBC count of 78 consecutive patients with newly diagnosed de novo AML. In all experiments, P value <0.05 has been considered as statistically significant.FITC-conjugated anti-CD31 induced increase in MFI of Cy-3 by 108.4% and we observed a CD31/CD38 cocapping. The inhibition of migration by anti-CD31 blocking mAb ranged from 2% to 66% of control and was correlated to the CD31 expression level (R=0.9236; P<0.0001), while migration in presence of anti-CD38 blocking mAb was about 80% of the control without correlation with CD38 expression level, suggesting a lesser role in the transendothelial migration process. The blasts exposed to ATRA exhibited a 1.3±0.1-fold increase in the CD38 expression (S/N of MFI) and a 40±10 % increase in adhesion to hyaluronate. The expression of CD31 and CD38 on the membrane of marrow leukemic cells of the AML patients were correlated (R=0.6621; P<0.0001). Moreover, peripheral WBC count was correlated with the CD31/CD38 ratio (R=0.5286; P=0.0129) and an excess of CD31 antigen was associated with an increased peripheral WBC count when comparing patients with ratio >1 to those with ratio <1 (P<0.0001). Interestingly, CD31/CD38 ratio was higher in AML (myelo)monocytic subtypes when comparing M4/M5 to all other FAB subtypes (1.2±0.1 and 0.7±0.1, respectively; P<0.0001).These findings suggest that extramedullar dissemination of AML cells depends on CD31 and CD38 coexpression level and give new insights into novel strategies to reduce the number of extramedullar tumor sites, notably in AML (myelo)monocytic subtypes.

Ligation of lymphocyte function-associated antigen-1 on monocytes decreases very late antigen-4-mediated adhesion through a reactive oxygen species-dependent pathway

AbstractMonocyte-endothelial adhesion plays an important role in monocyte trafficking and hence is important for immune responses and pathogenesis of inflammatory diseases including atherosclerosis. The cross-talk between different integrins on monocytes may be crucial for a coordinated regulation of the cellular adhesion during the complex process of transendothelial migration. By using monoclonal antibodies and recombinant intercellular adhesion molecule 1 (ICAM-1) to engage lymphocyte function-associated antigen 1 (LFA-1) on monocytic cells, we found that the cellular adhesion to vascular cell adhesion molecule 1 (VCAM-1) mediated by very late antigen 4 (VLA-4) was suppressed after this treatment and the suppression depended on the presence of reactive oxygen species (ROSs). Inhibition of production of ROSs through the use of inhibitor of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, but not inhibitors of mitochondrial electron transport chain or xanthine oxidase, revealed that this suppression on VLA-4-mediated cellular binding was mediated by ROSs produced by phagocyte NADPH oxidase. Activation of phosphoinositol-3 kinase and Akt appears to mediate this NADPH oxidase activation through p47phox phosphorylation and Rac-1 activation. Our results provide a novel pathway in which ROSs play a critical role in integrin cross-talk in monocytes. This signaling pathway may be important for cellular transition from firm arrest to diapedesis during monocyte trafficking. (Blood. 2004;104:4046-4053)