caa a22 4500 606221115 CHVBK 20210128101127.0 cr unu---uuuuu 210128e20150701xx s 000 0 eng 10.1007/s00380-014-0547-y doi (NATIONALLICENCE)springer-10.1007/s00380-014-0547-y Hepatocyte growth factor suppresses hypoxia/reoxygenation-induced XO activation in cardiac microvascular endothelial cells [Elektronische Daten] [Yingqian Zhang, Shunying Hu, Yundai Chen] 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 4h of hypoxia and followed by 1h 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. Springer Japan, 2014 Hepatocyte growth factor nationallicence Xanthine oxidase nationallicence Hypoxia/reoxygenation nationallicence Cardiac microvascular endothelia cells nationallicence Zhang Yingqian Department of Cardiology, Chinese PLA General Hospital, 28 Fuxing Rd, 100853, Beijing, China aut Hu Shunying Department of Cardiology, Chinese PLA General Hospital, 28 Fuxing Rd, 100853, Beijing, China aut Chen Yundai Department of Cardiology, Chinese PLA General Hospital, 28 Fuxing Rd, 100853, Beijing, China aut Heart and Vessels Springer Japan 30/4(2015-07-01), 534-544 0910-8327 30:4<534 2015 30 380 https://doi.org/10.1007/s00380-014-0547-y text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00380-014-0547-y text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Zhang Yingqian Department of Cardiology, Chinese PLA General Hospital, 28 Fuxing Rd, 100853, Beijing, China aut NATIONALLICENCE 700 1- Hu Shunying Department of Cardiology, Chinese PLA General Hospital, 28 Fuxing Rd, 100853, Beijing, China aut NATIONALLICENCE 700 1- Chen Yundai Department of Cardiology, Chinese PLA General Hospital, 28 Fuxing Rd, 100853, Beijing, China aut NATIONALLICENCE 773 0- Heart and Vessels Springer Japan 30/4(2015-07-01), 534-544 0910-8327 30:4<534 2015 30 380