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.

Protective effects of hepatocyte growth factor gene overexpression against hydrogen peroxide‐induced apoptosis in mesenchymal stem cells

“…It has also been reported that HGF has an anti‐inflammatory effect on the chronic kidney disease rat model . Interestingly, recent studies have shown that HGF protected cardiac microvascular endothelial cells against oxidative damage induced by hypoxia/reoxygenation . Based on these studies, we aimed to study the protective effect of HGF in MSCs exposed to harsh conditions.…”

“…7 Interestingly, recent studies have shown that HGF protected cardiac microvascular endothelial cells against oxidative damage induced by hypoxia/reoxygenation. 8 Based on these studies, we aimed to study the protective effect of HGF in MSCs exposed to harsh conditions. For this, we constructed a plasmid overexpressing HGF gene and investigated the effect of HGF-overexpression in MSCs.…”

Therapeutic Hypothermia Reduces Oxidative Damage and Alters Antioxidant Defenses after Cardiac Arrest

“…Despite the well-characterized increase in XO activity generating ROS during I/R injury [29], most studies are still trying to characterize the mechanisms of XO regulation, including the increase in XOR expression [2] and redox modifications due to I/R injury [6, 30]. In this respect, this is a pioneering study evaluating XO activity in post-CA patients who received MTH.…”

Microdialysis technique for in-vivo monitoring of •OH generation on myocardial injury in the rat

“…Elevation of intracellular Ca 2+ levels causes the conversion of xanthine dehydrogenase to XO [53,56], which produces superoxide. Superoxide, either directly or after conversion to •OH [57], damages the biological membranes [58] and other cellular components, including DNA [59], resulting in cell death.…”