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PubMed1990

Monoclonal antibody and synthetic peptide inhibitors of human tumor cell migration.

The processes of migration and invasion by human tumor cells are likely to involve specific cell surface receptors, such as receptors for the extracellular matrix molecules fibronectin, laminin, and collagen. We have examined the roles of several of these receptors using a set of monoclonal antibodies directed against the beta 1 integrin family, as well as a series of synthetic peptides reported to inhibit various interactions of each of these proteins with the cell surface. The most general inhibitor of tumor cell migration was found to be the anti-beta 1 monoclonal antibody 13, which inhibited the migration of human HT-1080 fibrosarcoma cells, 5637 bladder carcinoma cells, VA13 viral transformants, and HCT 116 colon carcinoma cells when fibronectin was the migration substrate. Moreover, this antibody was particularly effective in blocking cell migration on laminin, as well as migration within 3-dimensional collagen gels. It also inhibited in vitro invasiveness in a reconstituted basement membrane invasion assay (Matrigel assay) at concentrations as low as 1 microgram/ml. Integrins of the beta 1 class thus appear to play a central role in several types of migration by a variety of human tumor cell lines. Anti-alpha 5 fibronectin receptor monoclonal antibody 16 also significantly inhibited migration on fibronectin, but not on other substrates, in 3 of the 4 cell lines. Conversely, anti-alpha 2 monoclonal antibody F17 strikingly inhibited migration in 3-dimensional collagen gels, but not on other substrates, implicating the alpha 2 beta 1 integrin system in migration of tumor cells within collagenous matrices. A series of synthetic peptides previously reported to inhibit interactions of normal cells with fibronectin, laminin, and collagen were also tested as inhibitors of tumor cell migration. Peptides containing the Arg-Gly-Asp adhesive recognition signal were partially inhibitory, but with occasional exceptions, most other peptides had no effects on migration. Our results indicate the central importance of several specific beta 1 integrins in human tumor cell migration and show the effectiveness of monoclonal antibody treatment in blocking this process in vitro.

Amino Acid Sequence↗
PubMed1995

Epidermal growth factor stimulates integrin-mediated cell migration of cultured human corneal epithelial cells on fibronectin and arginine-glycine-aspartic acid peptide.

PURPOSE: The aim of this work was to show epidermal growth factor (EGF)-dependent migration of human corneal epithelial cells to fibronectin and GRGDSP peptide. The authors assessed the role of cell surface integrin heterodimer alpha 5 beta 1 in mediating haptotactic cell migration to fibronectin by the use of specific function-blocking integrin antibodies. METHODS: A haptotactic cell migration assay in a Boyden chamber was used to compare the relative migration of the cultured human corneal epithelial cells in the presence of fibronectin and GRGDSP peptide-coated filters. Epithelial cells were incubated in the presence of function-blocking integrin antibodies or anti-EGF-receptor antibodies to determine their role in haptotactic cell migration. RESULTS: Human corneal epithelial cells grown as primary cultures migrated in the presence of fibronectin or GRGDSP peptide, but only on stimulation with EGF. Antibodies to the EGF receptor blocked the EGF-mediated stimulation of haptotactic cell migration. Anti-beta 1 and anti-alpha 5 antibodies each inhibited haptotactic cell migration to fibronectin and GRGDSP peptide. CONCLUSIONS: Epidermal growth factor provides an important stimulus of haptotactic cell migration of human corneal epithelial cells. Stimulation of cell migration by EGF was maximal in the range of 5 to 10 ng/ml; this response was completely blocked by incubation with an anti-EGF receptor antibody. Function-blocking integrin antibodies, specifically anti-beta 1 and anti-alpha 5, inhibited integrin-mediated cell migration to fibronectin and GRGDSP peptide. These data suggest that EGF represents an essential initial stimulus for haptotactic cell migration of human corneal epithelial cells; furthermore, integrins are important in mediating cell migration to fibronectin and GRGDSP:

Amino Acid Sequence↗
PubMed1997

Migration in the urban-rural hierarchy of China: insights from the microdata of the 1987 National Survey.

Microdata from the 1987 National Population Survey are used to analyze internal migration in China in the 1980s. The focus is on the impact of migration policies on rural-urban migration. "There are two main findings. First, although the migration policy resulted in a very low migration level and systematic distortions in migration schedules, its encouragement of downward migrations was very ineffective, whereas its control on rural-to-urban migrations was partially weakened by the strong upward aspirations of rural families awakened by recent economic reform. Consequently, net in-migration contributed substantially to the growth both of city and of town populations. Second, although the level of education had a strong positive effect on the migration propensities both of males and of females in general, it had a strong negative effect on the migration propensities of females at the time of marriage, a finding which suggests that the families at subsistence income level tended to marry their daughters to grooms in other communities in order to reduce the risk of familial income shortfalls."

Asia↗
PubMed2004

Stent-graft migration: a reappraisal of analysis methods and proposed revised definition.

PURPOSE: To demonstrate the need for a radiographic definition of migration that accurately describes a specific failure mode of an aortic stent-graft. METHODS: The diagnosis of endograft migration, as defined by the Society for Vascular Surgery/American Association for Vascular Surgery (SVS/AAVS) standards, requires a synthesis of clinical and/or radiographic observations. Radiographic studies and clinical reports of 704 consecutive patients treated over a 6-year period with abdominal aortic endografts were retrospectively reviewed. According to the current SVS/AAVS standards, 25 patients were identified as having endograft migration. Follow-up computed tomographic scans and radiographs available for 24 of these patients were scrutinized from discharge to the time of any observed proximal or distal fixation system movement based upon the discharge or 30-day CT scan. Proximal migration was defined with respect to the origin of the superior mesenteric artery (SMA) as movement in a caudal or cranial direction of >/=10 mm or >2 times the reconstructed resolution of the imaging study (whichever measurement was less). Distal migration was similarly defined using the aortic bifurcation and respective hypogastric artery origin as reference points. In an effort to assess the scan-to-scan variation, the distance between the SMA and lowest renal artery, which was expected to remain consistent, was measured. RESULTS: Film analysis with application of the revised migration definition confirmed fixation system failure with respect to the native arterial system in 12 (50%) of the 24 patients. Subjects judged to have endograft migration according to the reporting standards but not to have radiographic evidence of migration based on the modified criteria included 2 proximal endoleaks without evidence of device movement treated with proximal extensions, 1 procedure-related migration, 2 type III endoleaks treated with a second prosthesis implanted within the first, 1 distal endoleak treated with a limb extension, 1 rupture with presumed distal limb migration, and 2 cases of component separation. Three limb extensions were placed in the absence of leak or migration. These 12 patients all had radiographic evidence that the proximal and distal aspects of the originally implanted device did not move with respect to the native arterial vasculature, thus confirming stability of the respective fixation system. CONCLUSIONS: Adherence to this comprehensive definition of device migration, which differs from the published reporting standards, allows differentiation of the specific mechanisms of device failure. Data viewed in this context will aid in the understanding of device strengths and weaknesses, potentially improve patient assessment, and encourage design modifications to address specific aspects relating to fixation failure.

Aorta, Abdominal↗
PubMed2001

Optimal prevention and management of proximal ureteral stent migration and remigration.

PURPOSE: We evaluated issues associated with proximal ureteral stent migration and remigration, including causes and management, and the predictability of ureteral length. MATERIALS AND METHODS: All proximal ureteral stent migrations that occurred from January 1997 to March 2000 were reviewed. Characteristics and treatment of the 33 patients with proximal ureteral stent migration were compared with those of 66 randomly selected controls who did not have stent migration. We also analyzed a subgroup of 6 cases of remigration. RESULTS: Of the ureteral stents 2% migrated proximally. Mean height was greater in patients with versus without a migrated stent (p = 0.028). The stent-to-ureter length ratio was lower in the migrated than in the nonmigrated group (p <0.0001). Patient height and side of migration were significant predictors of ureteral length (R2 = 0.3511, p <0.0001 and 0.0007, respectively). Of the patients who required continued ureteral stenting migrated stent management included placement of a longer stent in 9 (group 1) and a stent of equal length in 4 (group 2), and repositioning of the original stent in 4 (group 3). There was no remigration in group 1. However, migration recurred in 2 patients in group 2 (50%) and in all 4 in group 3 (100%). CONCLUSIONS: Proximal migration occurs when a stent is too short for the ureter. We recommend that ureteral length should be measured directly from an x-ray to select the optimal stent length. If it is necessary to continue stenting a ureter after migration has been detected, a longer stent should be placed.

Female↗
PubMed2006

Dominant-negative Rac increases both inherent and ionizing radiation-induced cell migration in C6 rat glioma cells.

Rho-like GTPases, including Cdc42, Rac1 and RhoA, regulate distinct actin cytoskeleton changes required for cell adhesion, migration and invasion. In the present study, we examined the role of Rac signaling in inherent migration, as well as radiation-induced migration, of rat glioma cells. Stable overexpression of dominant-negative Rac1N17 in a C6 rat glioma cell line (C6-RacN17) promoted cell migration, and ionizing radiation further increased this migration. Migration was accompanied by decreased expression of the focal adhesion molecules FAK and paxillin. Focal contacts and actin stress fibers were also reduced in C6-RacN17 cells. Downstream effectors of Rac include JNK and p38 MAP kinases. Irradiation transiently activated p38, JNK and ERK1/2 MAP kinases in C6-RacN17 cells, while p38 and JNK were constitutively activated in C6 control cells. Blocking JNK activity with JNK inhibitor SP600125 inhibited migration, suggesting that the JNK pathway may regulate radiation-induced, as well as inherent, migration of C6-RacN17 cells. Additionally, the radiation-induced migration increase was also inhibited by SB203580, a specific inhibitor of p38 MAP kinase. However, PD98059, a MEK kinase 1 inhibitor, failed to influence migration. This is the first evidence that suppression of Rac signaling may be involved in invasion or metastasis of glioma cells before and/or after radiotherapy. These data further suggest that radiotherapy for malignant glioma needs to be used with caution because of the potential for therapy-induced cell migration or invasion and that pharmacological inhibition of cell migration and invasion through targeting the Rac signaling pathway may represent a new approach for improving the therapeutic efficacy of radiotherapy for malignant glioma.

Animals↗
PubMed2004

Differential regulation of platelet-derived growth factor stimulated migration and proliferation in osteoblastic cells.

Osteoblastic migration and proliferation in response to growth factors are essential for skeletal development, bone remodeling, and fracture repair, as well as pathologic processes, such as metastasis. We studied migration in response to platelet-derived growth factor (PDGF, 10 ng/ml) in a wounding model. PDGF stimulated a twofold increase in migration of osteoblastic MC3T3-E1 cells and murine calvarial osteoblasts over 24-48 h. PDGF also stimulated a tenfold increase in 3H-thymidine (3H-TdR) incorporation in MC3T3-E1 cells. Migration and DNA replication, as measured by BrdU incorporation, could be stimulated in the same cell. Blocking DNA replication with aphidicolin did not reduce the distance migrated. To examine the role of mitogen-activated protein (MAP) kinases in migration and proliferation, we used specific inhibitors of p38 MAP kinase, extracellular signal regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). For these signaling studies, proliferation was measured by carboxyfluorescein diacetate succinimidyl ester (CFSE) using flow cytometry. Inhibition of the p38 MAP kinase pathway by SB203580 and SB202190 blocked PDGF-stimulated migration but had no effect on proliferation. Inhibition of the ERK pathway by PD98059 and U0126 inhibited proliferation but did not inhibit migration. Inhibition of JNK activity by SP600125 inhibited both migration and proliferation. Hence, the stimulation of migration and proliferation by PDGF occurred by both overlapping and independent pathways. The JNK pathway was involved in both migration and proliferation, whereas the p38 pathway was predominantly involved in migration and the ERK pathway predominantly involved in proliferation.

Animals↗
PubMed2002

p38 MAP kinase mediates platelet-derived growth factor-stimulated migration of hepatic myofibroblasts.

Although the migration of hepatic myofibroblasts (HMFs) contributes to the development of fibrosis, the signals regulating migration of these cells are poorly understood. In this study, we tested the hypothesis that HMF migration is stimulated by platelet-derived growth factor-BB (PDGF-BB) through p38 mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase (ERK) signaling pathways. This hypothesis was addressed by directly visualizing the migration of cultured human HMFs into a wound. PDGF-BB stimulated membrane ruffling, migration, and proliferation. PDGF-BB also induced activation of p38 MAP kinase, its downstream effector, heat shock protein (HSP) 27, ERK 1 and ERK 2, and p125 focal adhesion kinase (FAK). Selective antagonism of p38 MAP kinase blocked PDGF-BB-stimulated HSP 27 phosphorylation, membrane ruffling, and migration, but did not alter PDGF-BB-induced proliferation. Selective antagonism of ERK kinase inhibited PDGF-BB-induced ERK phosphorylation and proliferation, but did not affect PDGF-BB-stimulated migration. Concentrations of PDGF-BB that stimulated migration and proliferation did not influence myosin-dependent contractility. Neither selective inhibition of p38 MAP kinase nor ERKs altered PDGF-BB-induced activation of FAK. In conclusion, these results provide novel evidence indicating that (1) HMF migration is stimulated by PDGF-BB through the regulation of membrane ruffling by a p38 MAP kinase signaling pathway, (2) whereas p38 MAP kinase mediates PDGF-BB-stimulated migration, but not proliferation, ERKs mediate PDGF-induced proliferation, but not migration, and (3) increases in myosin-dependent contractility are not required for PDGF-BB-stimulated migration.

Becaplermin↗
PubMed2001

Both platelet-derived growth factor receptor (PDGFR)-alpha and PDGFR-beta promote murine fibroblast cell migration.

Cell motility plays a critical role for many physiological and pathological processes including wound healing, fibrosis, angiogenesis, and tumor metastasis. Platelet-derived growth factor (PDGF) is among the most potent stimuli for mesenchymal cell migration. The PDGF B-chain homodimer PDGF BB activates both alpha- and beta-receptor subunits (alpha-PDGFR and beta-PDGFR), and promotes cell migration in many cell types including fibroblasts and smooth muscle cells. PDGF-A chain homodimer PDGF AA activates alpha-PDGFR only, and its role for cell migration is still debatable. PDGF BB, but not PDGF AA, induces smooth muscle cell migration. Interestingly, alpha-PDGFR was shown to antagonize beta-PDGFR-induced smooth muscle cell migration. In the present study, we investigated the role of alpha-PDGFR and beta-PDGFR in PDGF-mediated cell migration of murine fibroblasts (NIH 3T3). Unlike smooth muscle cells, both PDGF AA and PDGF BB promoted NIH 3T3 cell migration. The effect of PDGF BB activation of beta-PDGFR alone for cell migration was examined using previously established NIH 3T3 clones in which alpha-PDGFR signaling is inhibited by a dominant-negative alpha-PDGFR, or an antisense construct of alpha-PDGFR. PDGF BB activation of beta-PDGFR alone was sufficient to induce cell migration, but the efficiency was significantly lower compared to PDGF activation of both receptors. These results showed that both alpha- and beta-PDGFRs promote fibroblast cell migration and their effects are additive. Taken together, we propose that cell-type specific alpha-PDGFR signaling is critical for regulation of mesenchymal cell migration in response to PDGF isoform, whereas beta-PDGFR mainly promotes cell migration.

3T3 Cells↗
PubMed1993

A major portion of polymorphonuclear leukocyte and T lymphocyte migration to arthritic joints in the rat is via LFA-1/MAC-1-independent mechanisms.

The importance of the leukocyte CD11/CD18 beta 2 integrins for leukocyte adhesion and for leukocyte migration to inflamed tissues is well recognized. However, the role of CD11/CD18 molecules and their subtypes, such as LFA-1 and MAC-1, in leukocyte migration to arthritis is not known. Here we report the effects of a new monoclonal antibody (mAb) TA-3, which recognizes and blocks rat LFA-1 (CD11a/CD18), on the migration of 51Cr-labeled rat blood polymorphonuclear leukocytes (PMNL) and 111In-labeled spleen T lymphocytes into the inflamed joints of rats with adjuvant arthritis and to dermal inflammation. The mAb MRC OX-42, which reacts with and blocks rat MAC-1 (CD11b/CD18), was also evaluated. In established arthritis, iv anti-LFA-1 (TA-3) inhibited, up to 50% (P < 0.006) of PMNL accumulation in the talar and metatarsal joints but had no effect on T lymphocyte migration to the joints. In the same animals, anti-LFA-1 partially (35-50%) inhibited PMNL accumulation in acute dermal inflammation induced by zymosan-activated serum (ZAS, C5ades Arg), IL-1 or endotoxin (P < 0.01) and suppressed T lymphocyte migration to dermal DTH, IFN-gamma, LPS, and poly-I:C-induced inflammation by 60-80%. Anti-MAC-1 (OX-42) by itself had no effect on PMNL migration. Anti-MAC-1 combined with anti-LFA-1 did not inhibit PMNL or T lymphocyte migration to joints any more than anti-LFA-1 alone. However, the addition of anti-MAC-1 to anti-LFA-1 further inhibited PMNL migration to acute dermal inflammation by up to 90%. These findings demonstrate that LFA-1 on PMNL plays a major role in PMNL migration but not in T cell migration to arthritic joints. MAC-1 on PMNL appears less important. Furthermore, while LFA-1 and MAC-1 on PMNL and LFA-1 on T lymphocytes mediate much of the migration to dermal inflammation, the migration of these cells to arthritis has a major LFA-1/MAC-1-independent component.

Animals↗
PubMed1999

Mature astrocytes transform into transitional radial glia within adult mouse neocortex that supports directed migration of transplanted immature neurons.

Neuronal migration is an essential step in normal mammalian neocortical development, and the expression of defined cellular and molecular signals within the developing cortical microenvironment is likely crucial to this process. Therapy via transplanted or manipulated endogenous precursors for diseases which involve neuronal loss may depend critically on whether newly incorporated cells can actively migrate to repopulate areas of neuronal loss within the adult brain. Previous studies demonstrated that embryonic neurons and multipotent precursors transplanted into the neocortex of adult mice undergoing targeted apoptosis of pyramidal neurons migrate long distances into neuron-deficient regions, undergo directed differentiation, accept afferent synaptic input, and make appropriate long-distance projections. The experiments presented here: (1) use time-lapse digital confocal imaging of neuronal migration in living slice cultures to assess cellular mechanisms utilized by immature neurons during such long distance migration, and (2) identify changes within the host cortical astroglial population that may contribute to this migration. Prelabeled embryonic day 17 mouse neocortical neurons were transplanted into adult mouse primary somatosensory cortex undergoing targeted apoptotic degeneration of callosal projection neurons. Four to 7 days following transplantation, living slice cultures containing the region of transplanted cells were prepared and observed. Sequential time-lapse images were recorded using a video-based digital confocal microscope. Transplanted cells displayed bipolar morphologies characteristic of migrating neuroblasts and moved in a saltatory manner with mean rates of up to 14 microm/h. To investigate whether a permissive glial phenotype may provide a potential substrate for this directed form of neuronal migration, slice cultures were immunostained with the RC2 monoclonal antibody, which identifies radial glia that act as a substrate for neuronal migration during corticogenesis. RC2 does not label mature stellate astrocytes, which express glial fibrillary acidic protein (GFAP). RC2 expression was observed in glial cells closely apposed to migrating donor neurons within the slice cultures. The timing and specificity of RC2 expression was examined immunocytochemically at various times following transplantation. RC2 immunostaining within regions of neuronal degeneration was transient, with peak staining between 3 and 7 days following transplantation. Strongly RC2-immunoreactive cells that did not express GFAP were found within these regions, but not in distant cortical regions or within control brains. RC2-positive cells were identified in recipient transgenic mice which express beta-galactosidase under a glial specific promoter. Coexpression of RC2 and beta-galactosidase identified these cells as host astroglia. These results demonstrate that adult cortical astrocytes retain the capacity to reexpress an earlier developmental phenotype that may partially underlie the observed active migration of transplanted neurons and neural precursors. Further understanding of these processes could allow directed migration of transplanted or endogenous precursors toward therapeutic cellular repopulation and complex circuit reconstruction in neocortex and other CNS regions.

Animals↗
PubMed1979

The mode of migration of neurons to the hippocampus: a Golgi and electron microscopic analysis in foetal rhesus monkey.

The mode of neuron migration from the site of their origin in the ventricular zone to area CA1 of the hippocampus was analysed with Golgi and electron microscopic methods during the first half of gestation in the foetal rhesus monkey. In the inner portion of the intermediate zone, the migrating cells have a bipolar form with one, or oaccasionally two, leading processes which do not reach the ammonic plate and with a single trailing process which usually ends within the intermediate zone. Both the nucleus and the cytoplasm of the migrating cells are relatively electron-dense and the latter contains organelles typical of young neurons as described in other brain regions. Analysis of electron micrographs from serial sections reveals that the length of the somata and of the leading and trailing processes of the migrating neurons is apposed to fascicles of radially oriented, electron-lucent, microtubule-filled fibres which are ultrastructurally similar to the radial glial fibres of the neocortex and to the Bergmann glial fibres of the cerebellum. The close (20 nm) apposition between the membranes of the migrating cell and the radial fibre is maintained even in areas where the fibres bend or curve tortuously. Migrating neurons situated at progressively more superficial levels of the intermediate zone become progressively more differentiated and complex. Thus, in the outer portion of the intermediate zone, the migrating cells acquire several additional cytoplasmic processes and occasionally a long thin axon-like process which courses into the incipient alveus. These cells have somewhat larger somata and less electron-dense nuclei and cytoplasm than the migrating neurons still situated in the inner part of the intermediate zone. Cells close to the ammonic plate usually have one to three cytoplasmic processes that enter the ammonic plate and terminate near their presumed final position. Migrating neurons situated at the lower border of the ammonic plate have a single large apical process which intermingles with neurons already in their final position and which sometimes traverses the ammonic plate. The apposition of the migrating neurons to the radial glial processes becomes less explicit as the cell soma enters the ammonic plate, reflecting the more complex three-dimensional intercellular relationships. However, the present analysis indicates that during the middle and late stages of neuronal migration to the hippocampus radial glial fibres may guide postmitotic young neurons across the intermediate zone to the ammonic plate in the same way that they guide neurons migrating to the superficial and middle layers of the neocortical plate.

Animals↗
PubMed1994

Endothelin-induced activation of neutrophil migration.

Migration of rabbit peritoneal neutrophils was stimulated by endothelin-1 (ET-1) up to 2.10(-8) M. Higher concentrations inhibited random migration. The stimulating effect of ET-1 was inhibited by BQ-123, a specific antagonist of the ETA receptor. A checkerboard assay showed that the stimulating effect of ET-1 on neutrophil migration was chemokinetic rather than chemotactic. Extracellular Ca2+ was required for the activating effect of ET-1. Non-selective calcium channel blockers such as econazole and La3+ strongly inhibited ET-1-activated migration but had little effect on fMLP-activated migration, underlining the importance of Ca2+ influx for ET-1-activated migration. Studies with electroporated neutrophils showed that the increase in migration was most pronounced at calcium concentrations between 100 nM and 1 microM. ET-1-activated migration of electroporated cells was completely blocked by low concentrations of calcium-channel blockers such as verapamil and nitrendipine. Migration by intact cells was inhibited by the same concentration of verapamil, but to a lesser degree; nitrendipine had little effect on migration of intact cells. This suggests that calcium derived from intracellular stores is required for migration activated by ET-1. Protein kinase C, protein tyrosine kinase, and phosphatase activity were involved in the activating effect of ET-1 on neutrophil migration. ET-1 did not induce exocytotic enzyme release, in neither the presence nor the absence of cytochalasin B.

Animals↗
PubMed1991

Fibronectin, hyaluronan, and a hyaluronan binding protein contribute to increased ductus arteriosus smooth muscle cell migration.

"Intimal cushions" which develop in the late gestation lamb ductus arteriosus (DA) are characterized by smooth muscle cells migrating into a large subendothelial space. Our previous in vitro studies, comparing DA cells with those from the aorta (Ao), have shown, even in early gestation, a 10-fold increase in DA endothelial incorporation of hyaluronan into the subendothelial matrix, a 2-fold increase in smooth muscle fibronectin synthesis and, in response to endothelial conditioned medium, a 2-fold increase in chondroitin sulfate. To determine whether these extracellular matrix components may be playing a role in inducing DA smooth muscle migration, we seeded Da or Ao smooth muscle cells onto three-dimensional collagen (2.0 mg/ml) gels and assessed migration 2, 5, and 8 days later. After 8 days, significantly greater numbers of DA compared to Ao cells were found invading the gels (23.1 +/- 3.1% vs 16.2 +/- 2.3%, P less than 0.01). Addition of GRGDS peptides (0.5 mM) or antibodies against fibronectin significantly decreased migration in the DA cells, but had no effect on migration in the Ao. Addition of endothelial conditioned medium to induce smooth muscle chondroitin sulfate production had no effect on DA cell migration. Inclusion of hyaluronan in the gel (0.5-1.5 mg), however, further enhanced DA cell migration, being greatest (31.9 +/- 3.1%) at a concentration of 1 mg/ml. Hyaluronan was without effect on Ao smooth muscle cell migration. The ability of hyaluronan to promote migration in cultures of DA smooth muscle cells was blocked completely by the addition of antibodies (1:100 dilution, 1 micrograms/ml) to a cell surface hyaluronan binding protein (HABP). As well, addition of anti-HABP to cells on gels containing collagen only significantly reduced migration in the DA but not the Ao. Immunofluorescent staining revealed that in DA cells, HABP was more concentrated in lamellipodia and leading edges than in Ao cells. As well, DA smooth muscle cells synthesized greater amounts of HABP as determined by Western immunoblotting and immunoprecipitation using polyclonal antisera to HABP. Thus, our studies indicate that both increased fibronectin and HABP contribute to the enhanced migration of DA smooth muscle cells. These results, together with our previous studies showing a 10-fold increase in hyaluronan accumulation in the DA endothelial matrix, would suggest a mechanism for increased DA smooth muscle migration into the subendothelial matrix observed in vivo.

Animals↗
PubMed1982

The effect of fibrin on endothelial cell migration in vitro.

Endothelial cells are known to migrate and come into contact with fibrin during numerous physiological processes, such as in wound healing and in tumor growth. The present study was initiated to investigate the effect of fibrin on endothelial cell migration in vitro. Endothelial cell migration was assayed by wounding confluent monolayers of bovine aortic endothelial cells with a razor blade and counting the number of cells crossing the wound per unit time. Wound-induced proliferation of endothelial cells was inhibited by mitomycin C-treatment without affecting endothelial cell migration, indicating that in this assay migration could be measured independent of proliferation. Migration of endothelial cells in vitro was inhibited by fibrin in a concentration dependent manner. Endothelial cell migration under fibrin was further reduced by plasminogen depletion of the serum, and fibrin still inhibited the migration of mitomycin C-treated endothelial cells. Kadish et al. (Tissue and Cell, 11, 99, 1979) previously reported that fibrin did not affect EC migration in vitro. The inability to inhibit EC migration with fibrin appears to be due to their assay system which employed agarose, since pre-treating the wounded monolayer with agarose eliminated the inhibition of EC migration by fibrin. The present results indicate that EC migration in vitro can be used as a model system for studying the interaction of fibrin with EC.

Animals↗
PubMed2004

Intracellular calcium transients are necessary for platelet-derived growth factor but not extracellular matrix protein-induced vascular smooth muscle cell migration.

PURPOSE: Vascular smooth muscle cell (SMC) migration is a critical component of the hyperplastic response that leads to recurrent stenosis after interventions to treat arterial occlusive disease. We investigated the relationship between intracellular calcium ([Ca(2+)](i)) and migration of vascular SMCs in response to platelet-derived growth factor (PDGF) and extracellular matrix (ECM) proteins. METHODS: Human saphenous vein SMCs were used for all experiments. SMC migration in response to agonists was measured with a microchemotaxis assay. A standard fluorimetric assay was used to assess changes in [Ca(2+)](i) in response to the various combinations of growth factors and ECM proteins. RESULTS: The calcium ionophore A23187 produced a rapid rise in [Ca(2+)](i) and a corresponding 60% increase in SMC migration, whereas chelation of [Ca(2+)](i) with BAPTA (1,2-bis [aminophenoxy] ethane-N,N,N',N'-tetraacetic acid) produced a fivefold decrease in PDGF-induced chemotaxis, suggesting that [Ca(2+)](i) is both sufficient and necessary for SMC migration. Stimulation of SMCs with PDGF produced an early peak followed by a late plateau in [Ca(2+)](i). To establish a relationship between temporal fluctuations in [Ca(2+)](i) and SMC migration, SMCs were pretreated with caffeine and ryanadine, which eliminated the initial peak but not the late plateau in [Ca(2+)](i), and had no effect on chemotaxis in response to PDGF. Incubation of SMCs with nickel chloride eliminated the late plateau, but had no effect on the initial peak in [Ca(2+)](i), and reduced PDGF-stimulated migration by fivefold. We then evaluated the role of calcium in SMC migration induced by ECM proteins such as laminin, fibronectin, and collagen types I and IV. All four matrix proteins stimulated SMC migration, but none produced an elevation in [Ca(2+)](i). Moreover, preincubation of SMCs with caffeine and ryanadine or nickel chloride had no effect on ECM protein-induced chemotaxis. CONCLUSION: [Ca(2+)](i) transients are necessary for PDGF but not ECM protein-induced SMC chemotaxis. Moreover, the ability of PDGF to stimulate vascular SMC migration appears dependent on influx of extracellular calcium through membrane channels. CLINICAL RELEVANCE: Recurrent stenosis after angioplasty or surgical bypass remains a significant challenge in treating vascular occlusive disease. In addition to growth factors, extracellular matrix (ECM) proteins may be potent agonists of this process. In this study we show that the influx of extracellular calcium is an important mechanism for platelet-derived growth factor-induced smooth muscle cell migration but not ECM-induced migration. Of note, in clinical trials calcium channel blockers failed to inhibit recurrent stenosis. Our data provide mechanistic insight to help explain this negative outcome in that therapies designed to inhibit restenosis depend on the effects of both growth factors and ECM proteins.

Calcimycin↗
PubMed2003

Nischarin inhibits Rac induced migration and invasion of epithelial cells by affecting signaling cascades involving PAK.

Nischarin, a cytosolic protein that binds the alpha5beta1 integrin, plays an important role in fibroblast migration, and in regulation of the actin cytoskeleton. The effect of Nischarin on Rac induced migration and invasion by breast and colon epithelial cell lines has been determined. In these cells, Rac potently induced migration, as well as invasion of matrix; both of these events were strongly inhibited by overexpression of Nischarin. To understand the mechanism of Nischarin's inhibitory role in Rac induced cell migration, several effector domain mutants of Rac1 were employed. Nischarin was able to inhibit migration induced by Rac effector mutants that can activate PAK and JNK, but not migration stimulated by other Rac mutants. Further, Nischarin inhibited PAK induced cell migration, while not affecting migration induced by MEKK1, a Rac effector in the JNK pathway. In addition, Nischarin failed to inhibit migration induced by MEK1, a downstream effector in the Ras-Raf-MEK-Erk signaling cascade. Furthermore, Nischarin does not affect Rac mediated JNK and PI3K activities. However, Rac induced migration and invasion were effectively blocked by pharmacological inhibitors of PI-3 kinase and MEK. These results suggest that several pathways contribute to cell migration, but that Nischarin selectively inhibits Rac driven signaling cascades that affect migration through PAK.

Animals↗
PubMed2006

In vitro assay of primary astrocyte migration as a tool to study Rho GTPase function in cell polarization.

Rho GTPases are key players in cell migration. The contribution of Rho, Rac, and Cdc42 to the regulation of the actin and microtubule cytoskeletons is essential for membrane protrusion and cell retraction (Etienne-Manneville and Hall, 2002). The polarization of these protrusive and retracting activities in a migrating cell is also under the control of Rho GTPases, in particular Cdc42 (Nobes and Hall, 1999). In vitro study of cell migration has shown that Cdc42 activity is required for polarized cell migration in several cell types, including fibroblasts, neutrophils, macrophages, and astrocytes (Allen et al., 1998; Etienne-Manneville, 2004; Etienne-Manneville and Hall, 2001; Palazzo et al., 2001; Srinivasan et al., 2003). Using scratch-induced migration assay, we have previously used primary astrocytes as a tool to study the molecular mechanisms controlling cell polarization at the onset of migration (Etienne-Manneville and Hall, 2001, 2003). On scratching of the monolayer, astrocytes polarize perpendicularly to the scratch to migrate and close the wound. Astrocyte polarization is characterized by the formation of a protrusion in the direction of migration, the elongation of the microtubules that fill the protrusion, and the reorientation of the centrosome, which serves as a microtubule-organizing center toward the direction of migration. This in vitro migration assay allows us to simultaneously investigate the mechanisms controlling cell migration, cell protrusion, and cell polarization. Primary astrocytes, although more constraining, provide a more physiological model than immortalized cell lines. Moreover, astrocyte culture can be obtained in a large number and, therefore, also allows biochemical analysis. Here I describe the procedure by which we can obtain and purify primary rat astrocytes and the different assays we have previously used to analyze the role of Rho GTPases and their downstream targets in cell migration and polarization.

Animals↗