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1 Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, PA, USA
2 Abramson Cancer Center Flow Cytometry and Cell Sorting Shared Resource and the Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, PA, USA
* To whom correspondence should be addressed. E-mail: abf{at}mail.med.upenn.edu.
Acute cessation of flow (ischemia) leads to depolarization of the endothelial cell membrane mediated by KATP channels and followed by production of reactive oxygen species (ROS) from NADPH oxidase. We postulated that ROS are a signal for initiating endothelial cell proliferation associated with loss of shear stress. Flow cytometry was used to identify proliferating CD31-positive pulmonary microvascular endothelial cells (MPMVEC) from wild-type, Kir6.2 -/-, and gp91phox -/- mice. MPMVEC were labeled with PKH26 and cultured in artificial capillaries for 72 hours at 5 dynes/cm2 (flow-adaptation) followed by 24 hours of stop flow or continued flow. ROS production during the first hour of ischemia was markedly diminished compared to wild-type in both types of gene-targeted MPMVEC. Cell proliferation was defined as the proliferation index (PI, proportion of cells divided). After 72 hours of flow, > 98% of PKH26 labeled wild-type MPMVEC were in a single peak (PI 1.0), and cells in the S+G2/M phases were 5.8% by cell cycle analysis. With ischemia (24 h), PI increased to 2.5 and cells in S+G2/M phases were 35%. Kir6.2 -/- and gp91phox -/- cells demonstrated PI <1.12 with 24 hours ischemia and diminished percent of cells in S+G2/M phases compared with wild-type. Catalase, DPI, a flavoprotein inhibitor, and cromakalim, a KATP channel agonist, markedly inhibited ROS production and cell proliferation in flow-adapted wild-type MPMVEC. These data indicate that absence of shear stress in flow-adapted MPMVEC results in mitosis associated with ROS generated by NADPH oxidase and requiring an intact cell membrane KATP channel.
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