Assessment of Rapid Morphological Changes Associated with Elevated cAMP Levels in Human Orbital Fibroblasts |
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Authors: | Lavanya Reddy Hwai-Shi Wang Charles R Keese Ivar Giaever Terry J Smith |
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Institution: | aSchool of Science, Rensselaer Polytechnic Institute and Applied BioPhysics, Inc. Troy, New York, 12180;bDivision of Molecular and Cellular Medicine, Department of Medicine, Department of Biochemistry and Molecular Biology, Albany Medical College and Samuel S. Stratton Veterans Affairs Medical Center, Albany, New York, 12208 |
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Abstract: | Orbital fibroblasts exhibit a phenotype distinct from that of other types of fibroblasts. Addition of prostaglandin E2(PGE2) to culture medium elicits a dramatic change in orbital fibroblast morphology. That response is mediated through the generation of cAMP. Orbital fibroblasts can generate high levels of PGE2through induction by proinflammatory cytokines of prostaglandin endoperoxide H synthase-2 (PGHS-2). Here we compare the influence on fibroblast morphology of exogenous PGE2, forskolin, and 8-br-cAMP to that mediated through PGHS-2 induction by a lymphocyte-derived cytokine. Within a few hours, orbital fibroblasts treated with any of these test compounds appear under phase-contrast microscopy to exhibit a stellate morphology. When these changes were assessed quantitatively by electric cell–substrate impedance sensing (ECIS), it became evident that 8-br-cAMP, forskolin, and PGE2initiated shape changes within 30 min of addition to the culture medium, while effects of the cytokine were first evident after approximately 3.5 h. Dermal fibroblasts failed to respond to any of these compounds with regard to changes in cellular morphology. Analysis of micromotion, manifested as small impedance fluctuations, revealed that orbital fibroblasts treated with 8-br-cAMP exhibit less motion than did untreated cells. These results suggest that orbital fibroblast shape can be altered by several compounds known to alter intracellular cAMP levels. They demonstrate the utility of ECIS in the assessment of very rapid and dynamic cellular events associated with changes in cell morphology. |
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Keywords: | Graves' disease thyroid associated ophthalmopathy electric cell&ndash substrate impedance sensing cytokine action |
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