Rapid plasticity of motor corticospinal system with robotic reach training.

Goal-directed reaching is important for the activities of daily living. Populations of neurons in the primary motor cortex that project to spinal motor circuits are known to represent the kinematics of reaching movements. We investigated whether repetitive practice of goal-directed reaching movements induces use-dependent plasticity of those kinematic characteristics, in a manner similar to finger movements, as had been shown previously. Transcranial magnetic stimulation (TMS) was used to evoke upper extremity movements while the forearm was resting in a robotic cradle. Plasticity was measured by the change in kinematics of these evoked movements following goal-directed reaching practice. Baseline direction of TMS-evoked arm movements was determined for each subject. Subjects then practiced three blocks of 160 goal-directed reaching movements in a direction opposite to the baseline direction (14 cm reach 180° from baseline direction) against a 75-Nm spring field. Changes in TMS-evoked whole arm movements were assessed after each practice block and after 5 min following the end of practice. Direction and the position of the point of peak velocity of TMS-evoked movements were significantly altered following training and at a 5-min interval following training, while amplitude did not show significant changes. This was accompanied by changes in the motor-evoked potentials (MEPs) of the shoulder and elbow agonist muscles that partly explained the change in direction, mainly by increase in agonist MEP, without significant changes in antagonists. These findings demonstrate that the arm representation accessible by motor cortical stimulation under goes rapid plasticity induced by goal-directed robotic reach training in healthy subjects.

Integrin alpha3beta1 engagement disrupts intercellular adhesion.

During tissue morphogenesis and tumor invasion, epithelial cells must undergo intercellular rearrangement in which cells are repositioned with respect to one another and the surrounding mesenchymal extracellular matrix. Using three-dimensional aggregates of squamous epithelial cells, we show that such intercellular rearrangements can be triggered by activation of beta1 integrins after their ligation with extracellular matrices. On nonadherent substrates, multicellular aggregates (MCAs) formed rapidly via E-cadherin junctional complexes and over time became compacted spheroids exhibiting a more epithelial phenotype. After MCAs were replated on culture substrates, the spheroids collapsed to yield tightly arranged cell monolayers. Cell-cell contact induced rapid elevation in E-cadherin levels, which was due to an increase in the metabolic stability of junctional receptors. During MCA remodeling of cell-cell adhesions, and monolayer formation, their E-cadherin levels fell rapidly. Similar behavior was obtained regardless of which ECM ligand-collagen type I, fibronectin, or laminin 1-MCAs were seeded on. In contrast, when seeded onto a matrix elaborated by squamous epithelial cells, cells in the MCA attached, spread, lost cell-cell junctions, and dispersed. Analysis identified laminin 5 as the active ECM ligand in this matrix, and MCA dispersion required functional beta1 integrin and specifically alpha3beta1. Furthermore, substrate-immobilized anti-integrin antibody effectively reproduced the epithelial-mesenchymal-like transition induced by the laminin 5 matrix. During the early stages of aggregate rearrangement and collapse, cells on laminin 5 substrates, but not those on collagen I substrates, exhibited intense cortical arrays of F-actin, microspikes, and fascin accumulation at their peripheral surfaces. These results suggest that engagement of specific integrin-ligand pairs regulates cadherin junctional adhesions during events common to epithelial morphogenesis and tumor invasion.

Radiation induced endothelial cell retraction in vitro: correlation with acute pulmonary edema.

We determined the effects of low dose radiation (<200 cGy) on the cell-cell integrity of confluent monolayers of pulmonary microvascular endothelial cells (PMEC). We observed dose- and time-dependent reversible radiation induced injuries to PMEC monolayers characterized by retraction (loss of cell-cell contact) mediated by cytoskeletal F-actin reorganization. Radiation induced reorganization of F-actin microfilament stress fibers was observed > or =30 minutes post irradiation and correlated positively with loss of cell-cell integrity. Cells of irradiated monolayers recovered to form contact inhibited monolayers > or =24 hours post irradiation; concomitantly, the depolymerized microfilaments organized to their pre-irradiated state as microfilament stress fibers arrayed parallel to the boundaries of adjacent contact-inhibited cells. Previous studies by other investigators have measured slight but significant increases in mouse lung wet weight >1 day post thoracic or whole body radiation (> or =500 cGy). Little or no data is available concerning time intervals <1 day post irradiation, possibly because of the presumption that edema is mediated, at least in part, by endothelial cell death or irreversible loss of barrier permeability functions which may only arise 1 day post irradiation. However, our in vitro data suggest that loss of endothelial barrier function may occur rapidly and at low dose levels (< or =200 cGy). Therefore, we determined radiation effects on lung wet weight and observed significant increases in wet weight (standardized per dry weight or per mouse weight) in < or =5 hours post thoracic exposure to 50 200 cGy x-radiation. We suggest that a single fraction of radiation even at low dose levels used in radiotherapy, may induce pulmonary edema by a reversible loss of endothelial cell-cell integrity and permeability barrier function.

E-cadherin regulates anchorage-independent growth and survival in oral squamous cell carcinoma cells.

Integrin-basement membrane interactions provide essential signals that promote survival and growth of epithelial cells, whereas loss of such adhesions triggers programmed cell death. We found that HSC-3 human squamous carcinoma cells survived and grew readily as monolayers, but when they were suspended as single cells, they ceased proliferating and entered into the apoptotic death pathway, characterized by DNA fragmentation. In contrast, if the suspended carcinoma cells were permitted to form E-cadherin-mediated multicellular aggregates, they not only survived but proliferated. However, aggregated normal keratinocytes were unable to survive in suspension culture and rapidly became apoptotic. Anchorage independence and resistance to apoptosis of HSC-3 cell aggregates required high levels of extracellular Ca2+ and was inhibited with function-perturbing anti-E-cadherin antibody. Resistance to suspension-induced apoptosis in cell aggregates paralleled the up-regulation of Bcl-2 but occurred in the absence of focal adhesion kinase activation. Analysis of suspension-induced death in a set of cloned squamous epithelial cell lines with different levels of E-cadherin expression revealed that receptor-positive cell clones evaded apoptosis and proliferated in three-dimensional aggregate culture, whereas cadherin-negative clones failed to survive. Collectively, these observations indicate that cadherin-mediated intercellular adhesions generate a compensatory mechanism that promotes anchorage-independent growth and suppresses apoptosis.

WR-1065 and radioprotection of vascular endothelial cells. II. Morphology.

Although the aminothiol WR-1065 protects normal tissues, its direct effect on the damage and restoration of the vascular endothelium is not clear. In endothelial cells, WR-1065 attenuates both the DNA damage and the G1-phase arrest induced by radiation. After the destruction of nearby endothelial cells, the survivors rearrange their cytoskeleton, migrate and replicate. To determine the effect of radiation on morphology and migration, portions of bovine aortic endothelial cell cultures were denuded with a pipette tip and irradiated (137Cs gamma rays). The following observations were noted after 5 Gy: within 10 min, there was increased formation of protein-mixed disulfides including actin-mixed disulfide; after 30-min, alpha 5 beta 1, the integrin receptor for fibronectin, was up-regulated on the apical membrane surface. Within 5 h, actin-containing stress fibers reorganized, although there was no change in the total filamentous (F-)actin content within the cells. Compared to controls after 24 h, the irradiated cells had migrated 15% farther (P < 0.01), and at the leading edge covered twice the surface area (P < 0.0001). The addition of 2 mM WR-1065 for 2 h before 5 Gy inhibited the increased expression of alpha 5 beta 1, promoted retention of stress fibers and prevented the enhanced cell migration and spreading. These results indicate that WR-1065 prevents radiation-induced morphological responses. This effect appears to be mediated by an impact on both adhesion molecule expression and cytoskeletal reorganization.

Inhibition of radiation-enhanced expression of integrin and metastatic potential in B16 melanoma cells by a lipoxygenase inhibitor.

Low-dose gamma radiation stimulates expression of phenotypic characteristics in B16 melanoma cells which regulate metastatic potential. A transient increase in the expression of an integrin receptor (alpha IIb beta 3) was observed after exposure of B16 melanoma cells to 0.25 to 2.0 Gy of gamma radiation. This increased receptor expression resulted in enhanced adhesion of tumor cells to fibronectin in vitro and increased experimentally induced metastasis in vivo. In this report, we determined a role for the 12-lipoxygenase metabolite, 12-HETE, in radiation-enhanced metastasis. A significant increase in biosynthesis of 12-HETE in B16 melanoma cells was detected < 5 min after exposure to 0.5 Gy gamma radiation. We then determined that radiation-enhanced expression of alpha IIb beta 3 integrin and adhesion of B16 melanoma cells to fibronectin in vitro and metastasis in vivo were reduced by treatment of the cells with the lipoxygenase inhibitor NDGA prior to irradiation. These findings suggest that low-dose radiation, at levels comparable to those used in fractionated or hyper-fractionated radiotherapy, increases the metastatic potential of surviving tumor cells via a rapid and transient alteration in lipoxygenase metabolism of arachidonic acid and surface expression of an integrin receptor.

Low dose radiation-induced endothelial cell retraction.

We characterized in vitro the effects of gamma-radiation (12.5-100 cGy) on pulmonary microvascular endothelial cell (PMEC) morphology and F-actin organization. Cellular retraction was documented by phase-contrast microscopy and the organization of actin microfilaments was determined by immunofluorescence. Characterization included radiation dose effects, their temporal duration and reversibility of the effects. A dose-dependent relationship between the level of exposure (12.5-100 cGy) and the rate and extent of endothelial retraction was observed. Moreover, analysis of radiation-induced depolymerization of F-actin microfilament stress fibres correlated positively with the changes in PMEC morphology. The depolymerization of the stress fibre bundles was dependent on radiation dose and time. Cells recovered from exposure to reform contact inhibited monolayers > or = 24 h post-irradiation. Concomitantly, the depolymerized microfilaments reorganized to their preirradiated state as microfilament stress fibres arrayed parallel to the boundaries of adjacent contact-inhibited cells. The data presented here are representative of a series of studies designed to characterize low-dose radiation effects on pulmonary microvascular endothelium. Our data suggest that post-irradiation lung injuries (e.g. oedema) may be induced with only a single fraction of therapeutic radiation, and thus microscopic oedema may initiate prior to the lethal effects of radiation on the microvascular endothelium, and much earlier than would be suggested by the time course for clinically-detectable oedema.