Mechanisms Of Transendothelial Migration Research Articles

Endothelial dysfunction of syphilis: Pathogenesis.

Treponema pallidum is the causative factor of syphilis, a sexually transmitted disease (STD) characterized by perivascular infiltration of inflammatory cells, vascular leakage, swelling and proliferation of endothelial cells (ECs). The endothelium lining blood and lymphatic vessels is a key barrier separating body fluids from host tissues and is a major target of T. pallidum. In this review, we focus on how T. pallidum establish intimate interactions with ECs, triggering endothelial dysfunction such as endothelial inflammation, abnormal repairment and damage of ECs. In addition, we summarize that migration and invasion of T. pallidum across vascular ECs may occur through two pathways. These two mechanisms of transendothelial migration are paracellular and cholesterol-dependent, respectively. Herein, clarifying the relationship between T. pallidum and endothelial dysfunction is of great significance to provide novel strategies for diagnosis and prevention of syphilis, and has a great potential prospect of clinical application.

Abstract 4112: Mechanisms of transendothelial migration by invasive breast carcinoma cells from patients

Abstract The majority of breast cancer related deaths are not due to the primary tumor, but rather to the dissemination of metastatic tumor cells from it to distant sites. Our lab has previously identified the tumor microenvironment of metastasis (TMEM) in mouse and human mammary tumors, sites where transendothelial migration, intravasation and dissemination occur. The constituent cells of TMEM are an endothelial cell, a perivascular TIE2-expressing macrophage (TEM) and an invasive Mena-over expressing tumor cell in direct contact. TMEM are present in human invasive breast tumors and the density of TMEM is positively associated with the risk of developing metastases. Using invasive ductal carcinoma cells of the breast obtained from patients by fine needle aspiration (FNA), we demonstrated that intravasation-directed transendothelial migration (iTEM) of these cancer cells requires macrophages and, depending on clinical subtype, involves either paracrine (macrophage CSF1-R; tumor cell EGFR), or both paracrine and autocrine (tumor cell EGFR + CSF1-R) signaling. Compared to the total population of primary breast cancer cells assayed, cells capable of transendothelial migration expressed relatively high MenaINV and low Mena11a levels, independently of clinical subtype. MenaINV and Mena11a are functionally distinct isoforms of Mena, a key regulator of motility and invasion. Depletion of MenaINV using siRNA showed that MenaINV is required for efficient macrophage-dependent iTEM of tumor cells. Furthermore, relative MenaINV expression correlated with the density of TMEM, which were previously shown to correlate with risk of metastasis in patients. From these observations we have identified the signaling pathways involved in macrophage-dependent tumor cell iTEM. Two novel small molecule inhibitors, Rebastinib (Tie2 inhibitor) and Altiratinib (c-MET inhibitor), have been identified that show great promise in preventing key steps in iTEM. Our data reveal targetable signaling events required for transendothelial migration of human breast cancer cells, and indicate that relative MenaINV levels and TMEM frequency are correlated prognostic markers of metastasis and therapeutic targets for most human breast cancers. Citation Format: Jeanine Pignatelli, Joan Jones, Xiaoming Chen, Bryan Smith, Daniel Flynn, John Condeelis, Maja Oktay. Mechanisms of transendothelial migration by invasive breast carcinoma cells from patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4112. doi:10.1158/1538-7445.AM2015-4112

Leukocyte transcellular diapedesis: Rap1b is in control

The neutrophil transmigration across the blood endothelial cell barrier represents the prerequisite step of innate inflammation. It is well known that neutrophils cross the endothelial barrier by transmigrating at the endothelial cell junction (‘paracellular’). However, in vivo and in vitro evidence have clearly demonstrated occurrence of an alternate mode of migration directly through the endothelial cell body (‘transcellular’). Despite our knowledge on mechanisms of transendothelial migration, it remains unclear which factors determine distinct modes of migration. We recently found that the Ras-like Rap1b GTPase limits neutrophil transcellular migration. Rap1b restrains transcellular migration by suppressing Akt-driven invasive protrusions while leaving the paracellular route unaffected. Furthermore, Rap1b limits neutrophil tissue infiltration in mice and prevents hyper susceptibility to endotoxin shock. These findings uncover a novel role for Rap1b in neutrophil migration and inflammation. Importantly, they offer emerging evidences that paracellular and transcellular migration of neutrophils are regulated by separate mechanisms. Here, we discuss the mechanisms of neutrophil transmigration and their clinical importance for vascular integrity and innate inflammation.

Abstract 3163: Transendothelial migration of uveal melanoma cells

Abstract Uveal melanoma, cancer arising from the pigmented uveal layer of the eye, is highly metastatic despite being limited to spreading only through the bloodstream. Almost half of patients develop distant metastatic disease, most often in the liver, even after the tumor-bearing eye is completely removed. The key to understanding and targeting uveal melanoma metastasis is in understanding how circulating tumors cells exit the bloodstream at distant sites of metastasis, and then invade and colonize distant organs. To address the first process, transendothelial migration, we modeled the blood vessel wall using primary human microvascular endothelial monolayers grown on polyacrylamide substrates that mimic the physiological stiffness of normal tissue. To these monolayers, we added 92.1 and OCM-1A uveal melanoma cells, and then collected time-lapse movies using spinning-disc confocal and differential interference contrast microscopy to follow the transmigration of the uveal melanoma cells. We found that both cell lines transmigrate through endothelial monolayers by a route distinctly different from the paracellular migration we see for trafficking immune cells. During this multistep process, uveal melanoma cells first identify paracellular junctions and then intercalate between endothelial cells, taking on a flattened morphology and maintaining contacts with their adjacent endothelial cells; reminiscent of vasculogenic mimicry. Tumor cells then extend invasive projections beneath adjacent endothelial cells, before eventually migrate under the endothelium. Immunofluorescence revealed cortical actin networks with strong cortactin staining within these invasive projections. To visualize these structures better, we transfected uveal melanoma cells with F-tractin, a fluorescent fusion protein that specifically binds filamentous actin without disrupting actin function, that allows imaging of actin dynamics in live cells. Time-lapse confocal images revealed complex actin-rich projections invading and sampling the interface between the endothelial monolayer and the substrate. Dynamic actin cytoskeletal reorganization was also apparent as cells migrated under the monolayer. Previous studies have linked inactivating mutations in BAP1 to metastatic spread of uveal melanoma in patients. To test whether BAP1 loss plays a role in transendothelial migration, we knocked down BAP1 expression in our uveal melanoma cell lines. We found that BAP1-depleted cells transmigrated significantly faster through endothelial monolayers in transwell assays than control knockdown cells. Time-lapse movies showed no significant differences in the first steps of initiation or intercalation between BAP1 knockdown and control cells. These studies identify a novel mechanism of transendothelial migration in uveal melanoma cells and suggest that BAP1 depletion specifically affects the ability of cells to invade under and exit the monolayer. Citation Format: Michael D. Onken, Olivia L. Mooren, Jinmei Li, John A. Cooper. Transendothelial migration of uveal melanoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3163. doi:10.1158/1538-7445.AM2014-3163

Cl– channels are expressed in human normal monocytes: a functional role in migration, adhesion and volume change

Increased adhesion and diapedesis of monocytes appear to be primary initiating factors in the pathophysiology of occlusive vascular diseases, including atherosclerosis and restenosis. However, the underlying mechanisms of transendothelial migration and invasion of monocytes into the blood vessels are not known. Alterations in ion channels on the cell membrane are generally involved in induced changes in shape and volume. In the present study, we investigated the expression and functional role of chloride channels in freshly isolated human blood monocytes. The Cl- currents in whole-cells were measured by the patch-clamp technique. We observed whole cell Cl- currents, which were time-independent and outwardly rectifying. The chloride channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and R(+)-[(6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5yl)-oxy]acetic acid 94 (IAA94) attenuated the Cl- currents. NPPB and IAA94 also inhibited chemotaxis of monocytes, as measured in Boyden chemotactic chambers, with the same sensitivity. NPPB but not IAA94, increased the cell volume as measured by shape change, and decreased tumour necrosis factor (TNF)-alpha-induced monocyte adhesion to endothelial cells. These results suggest that monocytes contain Cl- channels which regulate transendothelial migration of monocytes, due presumably to an alteration in cell volume.

Signal-regulatory protein alpha-CD47 interactions are required for the transmigration of monocytes across cerebral endothelium.

Monocyte infiltration into inflamed tissue requires their initial arrest onto the endothelial cells (ECs), followed by firm adhesion and subsequent transmigration. Although several pairs of adhesion molecules have been shown to play a role in the initial adhesion of monocytes to ECs, the mechanism of transendothelial migration is poorly defined. In this study, we have investigated the role of signal-regulatory protein (SIRP)alpha-CD47 interactions in monocyte transmigration across brain ECs. CD47 expression was observed in vivo on cerebral endothelium of both control animals and animals suffering from experimental allergic encephalomyelitis. To investigate whether SIRPalpha-CD47 interactions are instrumental in the trafficking of monocytes across cerebral EC monolayers, in vitro assays were conducted in which the migration of monocytes, but not adhesion, was found to be effectively diminished by blocking SIRPalpha and CD47 on monocytes and ECs, respectively. In this process, SIRPalpha was found to interact solely with its counterligand CD47 on ECs. Overexpression of the CD47 molecule on brain ECs significantly enhanced monocytic transmigration, but did not affect adhesion. SIRPalpha-CD47-mediated transendothelial migration involved Gi protein activity, a known signaling component of CD47. Finally, cross-linking of CD47 on brain ECs induced cytoskeletal reorganization of the endothelium, a process that was Gi protein independent. These data provide the first evidence that the interaction of CD47 with its monocytic counterligand SIRPalpha is of importance in the final step of monocyte trafficking into the brain, a critical event in the development of neuroinflammatory diseases.