, Volume 15, Issue 6, pp 693–704 | Cite as

Involvement of anion exchanger-2 in apoptosis of endothelial cells induced by high glucose through an mPTP-ROS-Caspase-3 dependent pathway

  • Qi-Ren Huang
  • Qing Li
  • Yuan-Hong Chen
  • Li Li
  • Li-Li Liu
  • Shui-Hong Lei
  • He-Ping Chen
  • Wei-Jie Peng
  • Ming He
Original Paper


Excess apoptosis of endothelial cells (EC) plays crucial roles in the onset and progression of vasculopathy in diabetes mellitus. Anion exchanger-2 (AE2) might be involved in the vasculopathy. However, little is known about the molecular mechanisms that AE2 mediated the apoptosis of EC. The purpose of this study was to explore the role of AE2 in the apoptosis of HUVECs induced by high glucose (HG) and its possible mechanisms. First, HUVECs were exposed to different glucose concentrations (5.5, 17.8, 35.6, 71.2 and 142.4 mmol/l, respectively, pH = 7.40) for different time points (12, 24, 48, 72, 120, and 168 h, respectively). Intracellular Cl concentration ([Cl]i), AE2 expression and the apoptosis were assayed. Then, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), Cl-free media or specific RNA interference (RNAi) for AE2 was used to confirm whether AE2 could mediate the apoptosis induced by HG. Finally, the mechanisms of the AE2-mediated apoptosis were investigated by detecting mitochondrial permeability transition pore (mPTP, ΔΨm) openings, reactive oxygen species (ROS) levels and Caspase-3 activity. We found that HG upregulated the AE2 expression and activity, increased [Cl]i and induced the apoptosis in a time- and concentration-dependent manner. The apoptosis of HUVECs by HG was possibly mediated by AE2 through an mPTP-ROS-Caspase-3 dependent pathway. These findings suggested that AE2 was likely to be a glucose-sensitive transmembrane transporter and a novel potential therapeutic target for diabetic vasculopathy.


Anion exchanger-2 Apoptosis Caspase-3 Endothelial cells Mitochondrial permeability transition pore Reactive oxygen species 



This work was supported by grants from the Natural Scientific Foundation of China (No. 30660058 and No. 30860111). We thank Dr. Huixin Deng, Xuan Jin, Shiwen Luo and Gregory D. Jensen for generous help in correcting the manuscript.


  1. 1.
    Ruderman NB, Williamson JR, Brownlee M (1992) Glucose and diabetic vascular disease. FASEB J 6:2905–2914PubMedGoogle Scholar
  2. 2.
    Pezet M, Verdetti J, Faury G (2004) Effect of glucose concentration on vascular function in aging: Action on calcium fluxes and vasomotricity induced by elastin peptides. J Soc Biol 198:279–286PubMedGoogle Scholar
  3. 3.
    The Diabetes Control and Complications Trial Research Group (1993) The effect of intensive treatment of diabetes on the development progression of long-term complications in insulin- dependent diabetes mellitus. N Engl J Med 329:977–986CrossRefGoogle Scholar
  4. 4.
    UK Prospective Diabetes Study Group (1998) Intensive blood–glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). Lancet 352:837–853CrossRefGoogle Scholar
  5. 5.
    Nakagami H, Kaneda Y, Ogihara T, Morishita R (2005) Endothelial dysfunction in hyperglycemia as a trigger of atherosclerosis. Curr Diabetes Rev 1:59–63CrossRefPubMedGoogle Scholar
  6. 6.
    Isermann B, Vinnikov IA, Madhusudhan T et al (2007) Activated protein C protects against diabetic nephropathy by inhibiting endothelial and podocyte apoptosis. Nat Med 13:1349–1358CrossRefPubMedGoogle Scholar
  7. 7.
    Nagaraj RH, Oya-Ito T, Bhat M, Liu B (2005) Dicarbonyl stress and apoptosis of vascular cells: prevention by alpha B-crystallin. Ann N Y Acad Sci 1043:158–165CrossRefPubMedGoogle Scholar
  8. 8.
    Detaille D, Guigas B, Chauvin C et al (2005) Metformin prevents high-glucose-induced endothelial cell death through a mitochondrial permeability transition-dependent process. Diabetes 54:2179–2187CrossRefPubMedGoogle Scholar
  9. 9.
    Sheu ML, Ho FM, Yang RS et al (2005) High glucose induces human endothelial cell apoptosis through a phosphoinositide 3-kinase-regulated cyclooxygenase-2 pathway. Arterioscler Thromb Vasc Biol 25:539–545CrossRefPubMedGoogle Scholar
  10. 10.
    Varma S, Lal BK, Zheng R et al (2005) Hyperglycemia alters PI3 K and Akt signaling and leads to endothelial cell proliferative dysfunction. Am J Physiol Heart Circ Physiol 289:H1744–H1751CrossRefPubMedGoogle Scholar
  11. 11.
    Ido Y, Carling D, Ruderman N (2002) Hyperglycemia-induced apoptosis in human umbilical vein endothelial cells: inhibition by the AMP-activated protein kinase activation. Diabetes 51:159–167CrossRefPubMedGoogle Scholar
  12. 12.
    Cheng G, Shao Z, Chaudhari B, Agrawal DK (2007) Involvement of chloride channels in TGF-B1-induced apoptosis of human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 293:L1339–L1347CrossRefPubMedGoogle Scholar
  13. 13.
    Lang F, Föller M, Lang K et al (2007) Cell volume regulatory ion channels in cell proliferation and cell death. Methods Enzymol 428:209–225CrossRefPubMedGoogle Scholar
  14. 14.
    Guan YY, Wang GL, Zhou JG (2006) The ClC-3 Cl-channel in cell volume regulation, proliferation and apoptosis in vascular smooth muscle cells. Trends Pharmacol Sci 27:290–296CrossRefPubMedGoogle Scholar
  15. 15.
    Shiio Y, Suh KS, Lee H, Yuspa SH, Eisenman RN, Aebersold R (2006) Quantitative proteomic analysis of myc-induced apoptosis: a direct role for Myc induction of the mitochondrial chloride ion channel, mtCLIC/CLIC4. J Biol Chem 281:2750–2756CrossRefPubMedGoogle Scholar
  16. 16.
    Lemonnier L, Shuba Y, Crepin A et al (2004) Bcl-2-dependent modulation of swelling-activated Cl- current and ClC-3 expression in human prostate cancer epithelial cells. Cancer Res 64:4841–4848CrossRefPubMedGoogle Scholar
  17. 17.
    Varela D, Simon F, Riveros A, Jørgensen F, Stutzin A (2004) NAD(P)H oxidase-derived H2O2 signals chloride channel activation in cell volume regulation and cell proliferation. J Biol Chem 279:13301–13304CrossRefPubMedGoogle Scholar
  18. 18.
    Shennan DB (2008) Swelling-induced taurine transport: relationship with chloride channels, anion-exchangers and other swelling-activated transport pathways. Cell Physiol Biochem 21:15–28CrossRefPubMedGoogle Scholar
  19. 19.
    Faber S, Lang HJ, Scholkens BA, Mutschler E (1998) Intracellular pH regulation in bovine aortic endothelial cells: evidence of both Na+/H+ exchange and Na+-dependent Cl-/HCO3-exchange. Cell Physiol Biochem 8:202–211CrossRefPubMedGoogle Scholar
  20. 20.
    Alper SL (2006) Molecular physiology of SLC4 anion exchangers. Exp Physiol 91:153–161CrossRefPubMedGoogle Scholar
  21. 21.
    Liu CJ, Hwang JM, Wu TT et al (2008) Anion exchanger inhibitor DIDS induces human poorly-differentiated malignant hepatocellular carcinoma HA22T cell apoptosis. Mol Cell Biochem 308:117–125CrossRefPubMedGoogle Scholar
  22. 22.
    Fujita H, Ishizaki Y, Yanagisawa A, Morita I, Murota SI, Ishikawa K (1999) Possible involvement of a chloride-bicarbonate exchanger in apoptosis of endothelial cells and cardiomyocytes. Cell Biol Int 23:241–249CrossRefPubMedGoogle Scholar
  23. 23.
    Huang Q, He M, Chen H et al (2007) Protective effects of sasanqua-saponin on injury of endothelial cells induced by anoxia and reoxygenation in vitro. Basic Clin Pharmacol Toxicol 101:301–308CrossRefPubMedGoogle Scholar
  24. 24.
    Reynolds A, Leake D, Boese Q et al (2004) Rational siRNA design for RNA interference. Nat Biotechnol 22:326–330CrossRefPubMedGoogle Scholar
  25. 25.
    Ui-Tei K, Naito Y, Takahashi F et al (2004) Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res 32:936–948CrossRefPubMedGoogle Scholar
  26. 26.
    Zang M, Gong J, Luo L et al (2008) Characterization of S338 phosphorylation for Raf-1 activation. J Biol Chem 283:31429–31437CrossRefPubMedGoogle Scholar
  27. 27.
    Lai ZF, Shao Z, Chen YZ, He M, Huang Q, Nishi K (2004) Effects of sasanquasaponin on ischemia and reperfusion injury in mouse hearts. J Pharmacol Sci 94:313–324CrossRefPubMedGoogle Scholar
  28. 28.
    Alvarez BV, Fujinaga J, Casey JR (2001) Molecular basis for angiotensin II-induced increase of chloride/bicarbonate exchange in the myocardium. Circ Res 89:1246–1253CrossRefPubMedGoogle Scholar
  29. 29.
    Mardones P, Medina JF, Elferink RP (2008) Activation of cyclic AMP signaling in Ae2-deficient mouse fibroblasts. J Biol Chem 283:12146–12153CrossRefPubMedGoogle Scholar
  30. 30.
    Ghio AJ, Grayck EN, Turi J et al (2003) Superoxide-dependent iron uptake: a new role for anion exchanger protein 2. Am J Respir Cell Mol Biol 29:653–660CrossRefPubMedGoogle Scholar
  31. 31.
    Stewart AK, Kurschat CE, Vaughan-Jones RD, Alper SL (2009) Putative re-entrant loop 1 of AE2 transmembrane domain has a major role in acute regulation of anion exchange by pH. J Biol Chem 284:6126–6139CrossRefPubMedGoogle Scholar
  32. 32.
    Humphreys BD, Jiang L, Chernova MN, Alper SL (1995) Hypertonic activation of AE2 anion exchanger in Xenopus oocytes via NHE-mediated intracellular alkalinization. Am J Physiol 268:C201–C209PubMedGoogle Scholar
  33. 33.
    Frische S, Zolotarev AS, Kim YH et al (2004) AE2 isoforms in rat kidney: immunohistochemical localization and regulation in response to chronic NH4Cl loading. Am J Physiol Renal Physiol 286:F1163–F1170CrossRefPubMedGoogle Scholar
  34. 34.
    Nickell WT, Kleene NK, Kleene SJ (2007) Mechanisms of neuronal chloride accumulation in intact mouse olfactory epithelium. J Physiol 583:1005–1020CrossRefPubMedGoogle Scholar
  35. 35.
    Alper SL (2009) Molecular physiology and genetics of Na+-independent SLC4 anion exchangers. J Exp Biol 212:1672–1678CrossRefPubMedGoogle Scholar
  36. 36.
    Chernova MN, Stewart AK, Jiang L, Friedman DJ, Kunes YZ, Alper SL (2003) Structure–function relationships of AE2 regulation by Ca(i)(2+)-sensitive stimulators NH(4+) and hypertonicity. Am J Physiol Cell Physiol 284:C1235–C1246PubMedGoogle Scholar
  37. 37.
    Stewart AK, Kurschat CE, Burns D, Banger N, Vaughan-Jones RD, Alper SL (2007) Transmembrane domain histidines contribute to regulation of AE2-mediated anion exchange by pH. Am J Physiol Cell Physiol 292:C909–C918CrossRefPubMedGoogle Scholar
  38. 38.
    Li Y, Wu H, Khardori R, Song YH, Lu YW, Geng YJ (2009) Insulin-like growth factor-1 receptor activation prevents high glucose-induced mitochondrial dysfunction, cytochrome-c release and apoptosis. Biochem Biophys Res Commun 384:259–264CrossRefPubMedGoogle Scholar
  39. 39.
    Chen G, Shen X, Yao J et al (2009) Ablation of NF-kappaB expression by small interference RNA prevents the dysfunction of human umbilical vein endothelial cells induced by high glucose. Endocrine 35:63–74CrossRefPubMedGoogle Scholar
  40. 40.
    Dong Z, Wang J, Zhong Q (2003) Postmitochondrial regulation of apoptosis by bicarbonate. Exp Cell Res 288:301–312CrossRefPubMedGoogle Scholar
  41. 41.
    Fujita H, Morita I, Murota S (2000) Hydrogen peroxide induced apoptosis of endothelial cells concomitantly with cycloheximide. J Atheroscler Thromb 7:209–215PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Qi-Ren Huang
    • 1
    • 2
  • Qing Li
    • 2
  • Yuan-Hong Chen
    • 2
  • Li Li
    • 2
  • Li-Li Liu
    • 2
  • Shui-Hong Lei
    • 3
  • He-Ping Chen
    • 2
  • Wei-Jie Peng
    • 1
    • 2
  • Ming He
    • 1
    • 2
  1. 1.Key State Laboratory of Food Science and TechnologyNanchang UniversityNanchangPeople’s Republic of China
  2. 2.Department of Pharmacology and Molecular TherapeuticsNanchang University School of Pharmaceutical ScienceNanchangPeople’s Republic of China
  3. 3.Department of Endocrinological and Metabolic Disease, The Second Affiliated HospitalNanchang UniversityNanchangPeople’s Republic of China

Personalised recommendations