Skip to main content
Log in

Hepatocyte growth factor suppresses hypoxia/reoxygenation-induced XO activation in cardiac microvascular endothelial cells

  • Original Article
  • Published:
Heart and Vessels Aims and scope Submit manuscript


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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others


  1. Beetsch JW, Park TS, Dugan LL, Shah AR, Gidday JM (1998) Xanthine oxidase-derived superoxide causes reoxygenation injury of ischemic cerebral endothelial cells. Brain Res 786:89–95

    Article  CAS  PubMed  Google Scholar 

  2. Ono T, Tsuruta R, Fujita M, Aki HS, Kutsuna S, Kawamura Y, Wakatsuki J, Aoki T, Kobayashi C, Kasaoka S, Maruyama I, Yuasa M, Maekawa T (2009) Xanthine oxidase is one of the major sources of superoxide anion radicals in blood after reperfusion in rats with forebrain ischemia/reperfusion. Brain Res 1305:158–167

    Article  CAS  PubMed  Google Scholar 

  3. Ali OS, Abdelgawad HM, Mohammed MS, El-Awady RR (2013) Ischemic heart diseases in Egypt: role of xanthine oxidase system and ischemia-modified albumin. Heart Vessels. doi:10.1007/s00380-00013-00413-00383

    PubMed  Google Scholar 

  4. Meneshian A, Bulkley GB (2002) The physiology of endothelial xanthine oxidase: from urate catabolism to reperfusion injury to inflammatory signal transduction. Microcirculation 9:161–175

    Article  CAS  PubMed  Google Scholar 

  5. Agarwal A, Banerjee A, Banerjee UC (2011) Xanthine oxidoreductase: a journey from purine metabolism to cardiovascular excitation-contraction coupling. Crit Rev Biotechnol 31:264–280

    Article  CAS  PubMed  Google Scholar 

  6. Hare JM, Berry CE (2004) Xanthine oxidoreductase and cardiovascular disease: molecular mechanisms and pathophysiological implications. J Physiol 555:589–606

    Article  PubMed Central  PubMed  Google Scholar 

  7. Wang X, Zhou Y, Kim HP, Song R, Zarnegar R, Ryter SW, Choi AMK (2004) Hepatocyte Growth Factor Protects against Hypoxia/Reoxygenation induced Apoptosis in Endothelial Cells. J Biol Chem 279:5237–5243

    Article  CAS  PubMed  Google Scholar 

  8. Date I, Takaga N, Takagi K, Kago T, Matsumoto K, Nakamura T, Takeo S (2004) Hepatocyte growth factor attenuates cerebral ischemia-induced learning dysfunction. Biochem Biophys Res Commun 319:1152–1158

    Article  CAS  PubMed  Google Scholar 

  9. Makiuchi A, Yamaura K, Mizuno S, Matsumoto K, Nakamura T, Amano J, Ito Ken-ichi (2007) Hepatocyte growth factor prevents pulmonary ischemia-reperfusion injury in mice. J Heart Lung Transplant 26:935–943

    Article  PubMed  Google Scholar 

  10. Guo Y, Su L, Li Y, Guo N, Xie L, Zhang D, Zhang X, Li H, Zhang G, Wang Y, Liu C (2013) The synergistic therapeutic effect of hepatocyte growth factor and granulocyte colony-stimulating factor on pulmonary hypertension in rats. Heart Vessels. doi:10.1007/s00380-013-0395-1

    Google Scholar 

  11. Li H, Yu H, Zhao J, Huang Y, Chen Q, Zhang N (2010) HGF suppresses high glucose-mediated oxidative stress in mesangial cells by activation of PKG and inhibition of PKA. Free Radic Biol Med 49:467–473

    Article  CAS  Google Scholar 

  12. Zhou YJ, Yang HW, Wang XG, Zhang H (2009) Hepatocyte growth factor prevents advanced glycation end products-induced injury and oxidative stress through a PI3 K/Akt-dependent pathway in human endothelial cells. Life Sci 85:670–677

    Article  CAS  PubMed  Google Scholar 

  13. Nishida M, Carley WW, Gerritsen ME, Ellingsen O, Kelly RA, Smith TW (1993) Isolation and characterization of human and rat cardiac microvascular endothelial cells. Am J Physiol Heart Circ Physiol 264:H639–H652

    CAS  Google Scholar 

  14. Zhang Z, Li W, Sun D, Zhao L, Zhang R, Wang Y, Zhou X, Wang H, Cao F (2011) Toll-like receptor 4 signaling in dysfunction of cardiac microvascular endothelial cells under hypoxia/reoxygenation. Inflamm Res 60:37–45

    Article  CAS  PubMed  Google Scholar 

  15. Yu G, Bolon M, Laird DW, Tyml K (2010) Hypoxia and reoxygenation-induced oxidant production increase in microvascular endothelial cells depends on connexin40. Free Radic Biol Med 49:1008–1013

    Article  CAS  PubMed  Google Scholar 

  16. Kong R, Jia G, Cheng ZX, Wang YW, Mu M, Wang XSJ, Pan SH, Gao Y, Jiang HC, Dl Dong, Sun B (2012) Dihydroartemisinin enhances Apo2L/TRAIL-mediated apoptosis in pancreatic cancer cells via ROS-mediated Up-regulation of death receptor 5. PLoS ONE 7:e37222

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  17. Ha Y, Dun Y, Thangaraju M, Duplantier J, Dong Z, Liu K, Ganapathy V, Smith SB (2011) Sigma receptor 1 modulates endoplasmic reticulum stress in retinal neurons. Invest Ophthalmol Vis Sci 52:527–540

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Przygodzkia T, Sokal A, Bryszewska M (2005) Calcium ionophore A23187 action on cardiac myocytes is accompanied by enhanced production of reactive oxygen species. Biochim Biophys Acta 1740:481–488

    Article  Google Scholar 

  19. Piekarska J, Szczypka M, Mrukowicz BO, Gorczykowski M (2009) Effect of phytohaemagglutinin-P on apoptosis and necrosis in Trichinella spiralis infected mice. Vet Parasitol 159:240–244

    Article  CAS  PubMed  Google Scholar 

  20. Hu R, Zhou P, Peng YB, Xu X, Ma J, Liu Q, Zhang L, Wen XD, Qi LW, Gao N, Li P (2012) 6-Shogaol induces apoptosis in human hepatocellular carcinoma cells and exhibits anti-tumor activity in vivo through endoplasmic reticulum stress. PLoS One 7:e39664

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. McNally JS, Saxena A, Cai H, Dikalov S, Harrison DG (2005) Regulation of xanthine oxidoreductase protein expression by hydrogen peroxide and calcium. Arterioscler Thromb Vasc Biol 25:1623–1628

    Article  CAS  PubMed  Google Scholar 

  22. Shida T, Nozawa T, Sobajima M, Ihori H, Matsuki A, Inoue H (2013) Fluvastatin-induced reduction of oxidative stress ameliorates diabetic cardiomyopathy in association with improving coronary microvasculature. Heart Vessels. doi:10.1007/s00380-013-0402-6

    PubMed  Google Scholar 

  23. Ozaki M, Haga S, Zhang HQ, Irani K, Suzuki S (2003) Inhibition of hypoxia/reoxygenation-induced oxidative stress in HGF-stimulated antiapoptotic signaling: role of PI3-K and Akt kinase upon rac1. Cell Death Differ 10:508–515

    Article  CAS  PubMed  Google Scholar 

  24. Wang G, Qian P, Jackson FR, Qiana G, Wu G (2008) Sequential activation of JAKs, STATs and xanthine dehydrogenase/oxidase by hypoxia in lung microvascular endothelial cells. Int J Biochem Cell Biol 40:461–470

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  25. Peng T, Yu G, Feng Q, Tyml K (2007) Abrupt reoxygenation of microvascular endothelial cells after hypoxia activates ERK1/2 and JNK1, leading to NADPH oxidase-dependent oxidant production. Microcirculation 14:125–136

    Article  PubMed  Google Scholar 

  26. Krause GS, White BC, Aust S, Nayini NR, Kumar K (1988) Brain cell death following ischemia and reperfusion: a proposed biochemical sequence. Crit Care Med 16:714–726

    Article  CAS  PubMed  Google Scholar 

  27. Kondapalli J, Loor G, Iwase H, Chandel NS, Waypa GB, Guzy RD, Hoek TLV, Schumacker PT (2011) Mitochondrial oxidant stress triggers cell death in simulated ischemia–reperfusion. Biochim Biophys Acta 1813:1382–1394

    Article  PubMed Central  PubMed  Google Scholar 

  28. Ogawa J, Saito S, Minamiya Y (2005) Pulmonary reexpansion causes xanthine oxidase-induced apoptosis in rat lung. Am J Physiol 289:L400–l406

    Article  Google Scholar 

  29. Laemmel E, Matharan ST, Duranteau J, Vicaut E (2004) Reoxygenation after hypoxia and glucose depletion causes reactive oxygen species production by mitochondria in HUVEC. Am J Physiol Heart Circ Physiol 287:R1037–R1043

    Google Scholar 

  30. Peters SC, Piper HM (2007) Reoxygenation-induced Ca2+ rise is mediated via Ca2+ influx and Ca2+ release from the endoplasmic reticulum in cardiac endothelial cells. Cardiovasc Res 73:164–171

    Article  CAS  PubMed  Google Scholar 

  31. Nanduri J, Vaddi DR, Khan SA, Wang N, Makerenko V, Prabhakar NR (2013) Xanthine oxidase mediates hypoxia-inducible factor-2a degradation by intermittent hypoxia. PLoS One 8:e75838

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  32. Lapatto R, Saksela M, Raivio KO (1999) Irreversible conversion of xanthine dehydrogenase to xanthine oxidase by a mitochondrial protease. FEBS Lett 433:117–120

    Google Scholar 

Download references


This work was supported by grants (81070185, 81102079) from the National Natural Science Foundation of China.

Conflict of interest

The authors declare no conflict of interest.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Yundai Chen.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, Y., Hu, S. & Chen, Y. Hepatocyte growth factor suppresses hypoxia/reoxygenation-induced XO activation in cardiac microvascular endothelial cells. Heart Vessels 30, 534–544 (2015).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: