title: Hepatocyte growth factor suppresses hypoxia/reoxygenation-induced XO activation in cardiac microvascular endothelial cells.
abstract: Hypoxia/reoxygenation (H/R) is one of the cellular stresses in pathological conditions, such as myocardial infarction, stroke and organ transplantation. Oxidative stress caused by reactive oxygen species (ROS) is a crucial element of H/R injury in vascular endothelial cells (ECs). Xanthine oxidase (XO) has been recognized to contribute to H/R injury. Of note, xanthine oxidoreductase is synthesized as xanthine dehydrogenase (XDH) and needs to be converted to XO to become a source of superoxide. Hepatocyte growth factor (HGF) has been found to protect ECs against H/R injury. The relation, however, between HGF and XO in ECs under H/R conditions remains to be determined. Primary cultured rat cardiac microvascular endothelial cells (CMECs) were exposed to 4 h of hypoxia and followed by 1 h of reoxygenation. Generation of ROS and cytosolic Ca2+ concentration was measured by flow cytometry qualification of DCFHDA and fluo-3 AM staining cells, respectively. XDH mRNA was qualified by qRT-PCR analysis. XO activity was determined by colorimetric assay and XO protein levels were determined by Western blot. Cell apoptosis was assessed by caspase-3 activity and Annexin V/PI staining. After H/R, cellular ROS production significantly increased. Both XO activity and XO protein increased after H/R. Cellular ROS elevation was inhibited by allopurinol (a potent XO inhibitor), indicting XO accounting for the generation of ROS after H/R. In addition, XDH mRNA increased after H/R, indicating a de novo XDH synthesis, which needs to be converted to XO to become a source of superoxide. Pretreatment of HGF inhibited the elevation of XO activity and XO protein level after H/R; however, HGF has no effect on the increase of XDH mRNA. We also find an increase of the cytosolic Ca2+ in CMECs after H/R. BAPTA-AM, a cell-permeable Ca2+ chelator, prevented the increase of XO activity and XO protein levels, implicating the elevated cytosolic Ca2+ concentration involvement in XO conversion and XO activation. HGF inhibited the elevation of cytosolic Ca2+ concentration in CMECs after H/R. Furthermore, HGF ameliorated H/R-induced CMECs apoptosis. These findings suggest a novel mechanism whereby HGF inhibited XO-generated ROS production after H/R treatment. H/R induces a de novo synthesis of XDH, the XO precursor. In addition, H/R increases cytosolic Ca2+ concentration and promotes a Ca2+ -involved XO conversion and XO activation. HGF has no effect on the increase of XDH mRNA; however, HGF inhibited the elevation of XO protein level and XO activity after H/R in the post-transcriptional level primarily by inhibiting the increase of cytosolic Ca2+ concentration. HGF protects CMECs from H/R-induced apoptosis by inhibiting the elevation of XO protein level and XO activity.
authors: Department of Cardiology, Chinese PLA General Hospital, 28 Fuxing Rd, Beijing, 100853, China.
authors: Yingqian
authors: Y
authors: Zhang
authors: Shunying
authors: S
authors: Hu
authors: Yundai
authors: Y
authors: Chen
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D015687:Cell Hypoxia; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology*; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects; D017228:Hepatocyte Growth Factor / Q000494:pharmacology*; D010100:Oxygen / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D014968:Xanthine Dehydrogenase / Q000378:metabolism*; D014969:Xanthine Oxidase / Q000378:metabolism*
chemical_list: D002614:Chelating Agents; D017382:Reactive Oxygen Species; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D017228:Hepatocyte Growth Factor; D014968:Xanthine Dehydrogenase; D014969:Xanthine Oxidase; D053148:Caspase 3; D010100:Oxygen