Biomedical subjects

L E Hinman

Publications and source records attributed to L E Hinman.

5 recordsLinked to original sources

PubMed1999

A wound-induced [Ca2+]i increase and its transcriptional activation of immediate early genes is important in the regulation of motility.

Upon mechanical wounding of a confluent quiescent monolayer, cells move into the denuded zone. However, it is not well known what signaling cascade connects release from contact inhibition to cell movement at the wound edge. Mechanical wounding induced an increase in the concentration of intracellular free Ca2+ ([Ca2+]i) in endothelial cells at the wound edge. The [Ca2+]i signal was required for the transcriptional activation of two immediate early genes (IEGs), c-fos and c-jun, since blocking Ca2+ influx with Gd3+ or EGTA reduced IEG transcription, while augmenting Ca2+ influx increased IEG transcription. The transcriptional activation of the IEGs depended on protein kinase C and calmodulin-dependent protein kinase since treatment with the inhibitors Calphostin C and KN-62 significantly reduced IEG expression. Briefly blocking Ca2+ influx also produced a long-term reduction of cell motility, while augmenting Ca2+ influx increased cell motility. To evaluate whether expression of IEGs might control cell movement, we microinjected sense or antisense cDNA to c-fos into cells after wounding. Antisense c-fos cDNA inhibited motility, while sense cDNA increased motility rates. These results suggested that the [Ca2+]i rise, induced by wounding, regulated the initiation of subsequent motility through the transcriptional activation of IEGs during wounding.

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PubMed1998

Intensity modulation of pseudocolor images.

Fura-2 is a popular real-time fluorescent indicator used to image intracellular, free calcium concentrations ([Ca2+]i) in living cells. One difficulty with fura-2-based digital imaging is how to best display the [Ca2+]i information that is acquired. Typically, only the [Ca2+]i as pseudocolor data are displayed, and the localization and intensity of the dye are not presented. This paper describes a technique that allows for the re-introduction of the intensity information. Recovery of the intensity information would de-emphasize regions with a poor signal-to-noise ratio and would more accurately report variations of sample morphology. A second advantage of this technique is that it requires no additional hardware since it is completely software based and can be run on all the major operating systems.

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PubMed1997

Wound-induced calcium waves in alveolar type II cells.

Alveolar type II epithelial (ATII) cells repopulate the alveolus after acute lung injury. We hypothesized that injury would initiate signals in nearby survivors. When rat ATII monolayers were wounded, elevations in intracellular free Ca2+ concentration ([Ca2+]i) began at the edge of the wound and propagated outward as a wave for at least 300 microns. The [Ca2+]i wave was due to both influx of extracellular Ca2+ and release of intracellular Ca2+ stores. Reducing Ca2+ influx with brief treatments of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid or Gd3+ reduced both the amplitude and the apparent speed. Draining intracellular Ca2+ stores by pretreatment with cyclopiazonic acid eliminated the [Ca2+]i wave. Therefore, the [Ca2+]i wave depended critically on intracellular Ca2+ stores. [Ca2+]i elevations propagated over a break in the monolayer, suggesting that extracellular pathways were involved. Furthermore, extracellular factors from injured cells elevated [Ca2+]i in uninjured cultures. We conclude that wounding produces a [Ca2+]i wave in surviving cells and part of this response is mediated by soluble factors released into the extracellular space during injury.

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PubMed1997

How do injured cells communicate with the surviving cell monolayer?

Mechanically scratching cell monolayers relieves contact inhibition and induces surviving cells near the wound edge to move and proliferate. The present work was designed to test whether surviving cells passively respond to newly available space, or whether cells are actively stimulated by signals from injured cells nearby. We monitored intracellular free Ca2+ ([Ca2+]i) while scratching confluent monolayers of bovine pulmonary endothelial cells and mouse mammary epithelial cells. Within seconds after wounding, a transient elevation of [Ca2+]i was observed in surviving cells. In endothelial cells, the [Ca2+]i elevation propagated into the monolayer for a distance of 10 to 12 cell rows at a speed of 20 to 28 microm/second. The amplitude of the wave of [Ca2+]i was reduced as it propagated into the monolayer, but the velocity of the wave was nearly constant. Cells that experienced the [Ca2+]i elevation had intact plasma membranes, and survived for over 24 hours post wounding. Removing extracellular Ca2+ decreased the amplitude by two-thirds and reduced the propagation rate by half, suggesting that Ca2+ influx contributed to the increased [Ca2+]i. To determine how [Ca2+]i waves were stimulated, we blocked extracellular communication by fluid perfusion or intercellular communication by breaks in the monolayer. In bovine pulmonary artery endothelial cultures, the [Ca2+]i wave passed over breaks in the monolayer, and was prevented from traveling upstream in a perfusion chamber. Conditioned media from injured cells also elevated [Ca2+]i in unwounded reporter cultures. In mouse mammary epithelial monolayers with established cell-cell contacts, the [Ca2+]i wave passed over breaks in the monolayer, but was only partially prevented from traveling upstream during perfusion. These experiments showed that mechanical wounds lead to long distance, [Ca2+]i-dependent communication between the injured cells and the surviving cell monolayer through at least two mechanisms: first, extracellular release of a chemical stimulus from wounded cells that diffused to neighboring cells (present in both monolayers); second, transmission of an intercellular signal through cell-cell junctions (present in the mammary epithelial monolayers). Thus, mechanical injury provided a direct, chemical stimulus to nearby cells which have not themselves been damaged.

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PubMedDate not supplied

Co-ordination between localized wound-induced Ca2+ signals and pre-wound serum signals is required for proliferation after mechanical injury.

The signals which initiate proliferation of endothelial cells after injury are important for selective blood vessel growth during wound healing or tumour growth. Upon mechanically wounding quiescent cells, a transient [Ca2+]i increase was induced in cells at the wound edge. These same cells proliferated 18-24 h post wounding, as measured by bromodeoxyuridine incorporation. The localized Ca2+ signal was required specifically during wounding since blocking Ca2+ influx reduced proliferation by 40-50%. Proliferation also required serum since starvation reduced proliferation by 80%. Serum-starved cells proliferated if briefly primed with serum prior to wounding. The signals derived from serum and [Ca2+]i combined at least additively to induce proliferation. Therefore, serum priming followed by a single, transient Ca2+ signal induced by mechanical injury must occur in a temporally and spatially regulated manner for normal proliferation. Co-ordination between signalling cascades induced by growth factors and release from contact inhibition might be obligatory for localized re-endothelialization after injury.

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