title: Endothelial cell proliferation associated with abrupt reduction in shear stress is dependent on reactive oxygen species.
abstract: We have shown previously that flow-adapted endothelial cells respond to cessation of flow with cell membrane depolarization and increased production of reactive oxygen species, resulting in activation of transcription factors and increased DNA synthesis. This study utilized flow cytometry to evaluate cellular proliferation with ischemia and to determine the role of reactive oxygen species and apoptosis. PKH26-labeled rat pulmonary microvascular endothelial cells were seeded in an artificial capillary system and subjected to flow at 5 dynes/cm(2) for 96 h or for 72 h followed by 24 h of simulated "ischemia." Ischemia resulted in a 2.5-fold increase in the cellular proliferation index. Cell-cycle analysis showed G0/G1 arrest and decreased S plus G2/M during flow adaptation, whereas ischemia resulted in a three-fold increase of cells in S plus G2/M phases. Apoptotic cells as detected by TUNEL and annexin V binding assays were ~5% of total cells with no differences between static, flow-adapted, and "ischemic" groups. Reactive oxygen species production during a 1-h period following onset of ischemia was confirmed by oxidation of the fluorophore, dichlorofluorescein, and was inhibited by cromakalim, a K(ATP) channel agonist, or diphenyleneiodonium, a flavoprotein inhibitor. Cromakalim and diphenyleneiodonium also markedly inhibited cell proliferation in the flow-adapted ischemic cells, but had no effect on subconfluent cells cultured under static conditions. These results indicate reactive oxygen species-dependent endothelial cell proliferation in flow-adapted microvascular endothelial cells as a response to ischemia and indicate that this response is not a consequence of apoptosis.
authors.affiliation: Institute For Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104-6068, USA.
authors.forename: Tatyana
authors.initials: T
authors.lastname: Milovanova
authors.forename: Yefim
authors.initials: Y
authors.lastname: Manevich
authors.forename: Alex
authors.initials: A
authors.lastname: Haddad
authors.forename: Shampa
authors.initials: S
authors.lastname: Chatterjee
authors.forename: Jonni S
authors.initials: JS
authors.lastname: Moore
authors.forename: Aron B
authors.initials: AB
authors.lastname: Fisher
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002453:Cell Cycle / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D019806:Cromakalim / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D020287:In Situ Nick-End Labeling; D007511:Ischemia / Q000378:metabolism; D008833:Microcirculation; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013314:Stress, Mechanical; D014665:Vasodilator Agents / Q000494:pharmacology
chemical_list: D017382:Reactive Oxygen Species; D014665:Vasodilator Agents; D019806:Cromakalim