Effect of erythropoietin on endothelial cell apoptosis induced by high glucose

Diabetes Research and Clinical Practice 66S (2004) S103–S107

Effect of erythropoietin on endothelial cell apoptosis induced by high glucose Naotaka Sekiguchi∗ , Toyoshi Inoguchi, Kunihisa Kobayashi, Hajime Nawata Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan Received 28 August 2003; accepted 25 September 2003

Abstract Erythropoietin (Epo) has been reported to inhibit apoptosis of neuron and erythroid cells. In this study, we examined an effect of high glucose on apoptosis of endothelial cells and investigated an anti-apoptotic effect of Epo. Human aortic endothelial cells were incubated with normal or high glucose for 72 h, and apoptotic cells were detected by TUNEL assay. Simultaneously, Epo (100 U/ml) was added to the high glucose medium to examine an inhibitory effect on the apoptosis induced by high glucose. Activity of caspase-3 was also measured using a specific substrate. To investigate a possible mechanism of Epo’s action on apoptosis, phosphorylation of Akt was examined by applying Epo. Incubation with high glucose increased apoptosis of endothelial cells, whereas this effect was prevented by co-incubation with Epo. Caspase-3 activity was also increased (1.4-fold) by incubation with high glucose, and the activation of caspase-3 was normalized to the control level by co-incubation with Epo. Furthermore, Epo-induced phosphorylation of Akt in dose-dependent manner. In conclusion, we demonstrated that incubation with high glucose activated caspase-3 and induced apoptosis of endothelial cells. Epo was shown to phosphorylate Akt, leading to the inhibition of caspase-3 activation and apoptosis induced by high glucose. These results suggest that reduced production of Epo in patients with end-stage of nephropathy may accelerate diabetic angiopathy and that replacing therapy with Epo might inhibit endothelial cell apoptosis and diabetic angiopathy. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Erythropoietin; Apoptosis; Endothelial cell; Diabetic nephropathy

1. Introduction Apoptosis is related to the onset and development of diabetes. It has been reported that high glucose [1], free fatty acid [2], oxidative stress [3], c-Myc [4] and ∗ Corresponding author. Tel.: +92 642 5284; fax: +92 642 5287. E-mail address: [email protected] (N. Sekiguchi).

Fas [5] induce apoptosis of pancreatic ␤ cells, leading to the development of diabetes. Also, high glucose, AGE, glycated LDL and oxidative stress have been shown to induce cell apoptosis in retina [6], kidney, neuron, myocardial cells and vascular endothelial cells, which is related to the acceleration of diabetic complications and atherosclerosis [7–14]. Erythropoietin (Epo) is produced mainly in kidney and regulates red cell production. It binds to a cell surface receptor and leads to intracellular activation

0168-8227/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2004.05.007

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of several kinase pathways. Recently, Epo has been reported to inhibit apoptosis of neuron and erythroid cells induced by ischemia and metabolic abnormalities [15,16]. Clinically, it is often seen that atherosclerosis accelerates in patients with end-stage of diabetic nephropathy in which the production of Epo is reduced. Since Epo’s receptors are present on the membrane of vascular endothelial cells, it is possible that decreased production of Epo might induce apoptosis of vascular endothelial cell, which leads to the acceleration of atherosclerosis. Therefore, in this study, we examined an effect of high glucose on apoptosis of vascular endothelial cells and investigated an anti-apoptotic effect and possible mechanisms of Epo.

2. Materials and methods 2.1. Cell culture and treatments Human aortic endothelial cells were purchased from Clonetics. The endothelial cells were cultured in endothelial cell basal medium (EBM; Cambrex, Walkersville, MD) supplemented with hFGF-B, VEGF, IGF-1, ascorbic acid, hEGF, hydrocortisone and 20% fetal bovine serum (FBS). The cells from second to seventh passages were transferred on chamber slides or culture dishes and incubated with normal (100 mg/dl) or high (450 mg/dl) glucose with/without an application of Epo (Chugai Pharmacological Corp., 100 U/ml). Apoptosis of the endothelial cells was evaluated with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Caspase-3 activities were also measured using a specific fluorescent substrate as follows. 2.2. Evaluation of apoptotic cells; TUNEL Assay Apoptosis was evaluated by TUNEL assay [17,18]. Human aortic endothelial cells were cultured with normal (100 mg/dl) or high (450 mg/dl) glucose with/without Epo (100 U/ml) on chamber slides and were fixed in 1% paraformaldehyde in PBS. Then, the slides were processed for a TUNEL assay. An ApopTag in situ detection kit from Serologicals Corporation (Norcross, GA) was used according to the

manufacturer’s instructions. Briefly, the slides were treated with H2 O2 and incubated with the reaction mixture containing TdT and digoxigenin-conjugated dUTP for 1 h at 37 ◦ C. Labeled DNA was visualized with peroxidase-conjugated anti-digoxigenin antibody using 3,3 -diaminobenzidine (DAB) as the chromogen. After washing in dH2 O, the cells were counterstained in 0.5% methyl green, and the specimens were mounted with coverslips. Then, 100 of endothelial cells were counted under a microscope, and the percentages of brown colored TUNEL-positive cells were quantified. 2.3. Measurement of caspase-3 Activity Caspase-3 activity assay kit from Oncogene Research Products (Boston, MA) was used according to the manufacturer’s instructions [19,20]. Briefly, after reaching confluent on 96-well-plate, the endothelial cells were incubated with normal (100 mg/dl) or high (450 mg/dl) glucose with/without Epo (100 U/ml) for 72 h in accordance with TUNEL assay. Assay buffer containing 10 mM DTT was applied on the cells, followed by an addition of caspase-3 fluorescent substrate conjugate, which is DEVD (Asp-Glu-Val-Asp) peptide labeled with fluorescent 7-amino-4-trifluoromethyl coumarin (AFC). The plate was covered and incubated at 37 ◦ C for 2 h, and fluorescence of 400 nm excitation and 505 nm emission were read using a fluorescent plate reader. 2.4. Detection of phosphorylated Akt To investigate a possible mechanism of Epo’s action, Akt phosphorylation was examined by Western blot analysis. Endothelial cells were plated on 6-well-plate and incubated until the cells reached confluent on the well. The cells were exposed to Epo (0.1–100 U/ml) for 0–30 min. After the incubation with Epo, the cells were lysed using RIPA buffer containing NP-40, sodium deoxycholate, SDS, PMSF, aprotinin, leupeptin, sodium orthovanadate and sodium fluoride (Sigma). The protein concentration was assayed with an aliquot of each, 40 ␮g of protein was boiled in sample buffer. The protein separated via SDS-PAGE and subsequently transferred to nitrocellulose filters for Western blotting. Filters were probed using anti-phosphoAkt antibodies (Santa Cruz),

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followed by exposing to HRP-conjugated anti-rabbit secondary antibodies (Amersham). Antigen–antibody complexes were then visualized by enhanced chemiluminescence (ECL). The intensity was measured and quantitated. 2.5. Inhibition of phosphatidylinositol-3 (PI-3) kinase pathway To determine whether Epo plays an anti-apoptotic effect via PI-3 kinase pathway, PI-3 kinase inhibitors, wortmannnin (200 nm) or LY294002 (20 ␮M), were applied on endothelial cells in the presence of Epo. The cells were incubated with high glucose medium containing 100 U/ml of Epo with/without wortmannin or LY294002. The effects of PI-3 kinase inhibitors on cell apoptosis were examined by TUNEL assay, and activities of caspase-3 were also measured as well. 2.6. Statistical analysis Data were collected from repeated experiments and are presented as mean ± S.D. One-way ANOVA was used for statistical analysis. Differences were considered to be significant at P < 0.05. 3. Results 3.1. TUNEL assay We determined the effect of high glucose on apoptosis in cultured endothelial cells by TUNEL assay. As expressed in the Section 2, the apoptosis of endothelial cells was expressed quantitatively as a percentage of TUNEL positive cells. Compared with normal level of glucose (100 mg/dl), high glucose (450 mg/dl) culture for 72 h induced a significant increase in the number of brown colored TUNEL-positive cells (glucose 100 mg/dl; 0.7 ± 0.5% versus glucose 450 mg/dl; 4.3 ± 0.5%, P < 0.05). However, when Epo (100 U/ml) was added to high glucose medium, the number of the apoptotic cells was significantly decreased (glucose 450 mg/dl + Epo 100 U/ml; 1.3 ± 0.5%, P < 0.05).

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effectors such as caspases-3, -6 and -7. Among these caspases, caspase-3 is considered as a key enzyme in the pathogenesis of cell apoptosis, we measured caspase-3 activities in parallel with TUNEL assay. In consistent with the result of TUNEL assay, high glucose incubation for 72 h caused caspase-3 activation in endothelial cells. As compared with the activity in the cells incubated with normal glucose, caspase-3 activity was increased by 1.46-fold (P < 0.05) in the cells incubated with high glucose. On the contrary, application of 100 U/ml Epo to high glucose medium significantly ameliorated the caspase-3 activation to the control level (P < 0.05). 3.3. Phosphorylation of Akt In neuron and erythroid cells, Epo has been reported to activate PI-3 kinase and phosphorylate Akt and act against cell apoptosis. Therefore, we examined an effect of Epo on Akt phosphorylation in endothelial cells. Time response curve shows that Epo induced Akt phosphorylation in endothelial cells, maximum at 10 min after the exposure. Also, Epo was shown to phosphoryle Akt in dose dependent manner, maximum at 10 min after exposure of 100 U/ml Epo. 3.4. Inhibition of PI-3 kinase pathway According to the result that Epo induced phosphorylation of Akt, it is suggested that Epo may play an anti-apoptotic effect through PI-3 kinase pathway. To confirm the possibility, we attempted to block PI-3 pathway using its inhibitors, wortmannin (200 nM) or LY294002 (20 ␮M) in the presence of Epo. TUNEL assay revealed that inhibition of PI-3 pathway by wortmannin or LY294002 eliminated the anti-apoptotic effect of Epo. Similarly, caspase-3 activity was increased to the level of high glucose culture by the application of PI-3 kinase inhibitors. These results indicate that Epo possesses an anti-apoptotic effect through PI-3 kinase pathway.

3.2. Caspase-3 activity

4. Discussion

Caspases are separated into two subsets; the initiator caspases such as caspases-8 and -9, and the

Endothelial dysfunction induced by diabetes is a key component in the pathophysiology of cardiovas-

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cular abnormalities. Actually, cell death induced by high glucose has been shown in endothelial cells in vitro [10,11,21], and we also observed the apoptotic phenomenon by high glucose culture in human aortic endothelial cells. Several mechanisms of endothelial dysfunction in diabetes mellitus have been proposed so far, but one possibility is increased reactive oxygen species (ROS), because addition of antioxidants to cultured endothelial cells suppressed the activation of caspase-3 and the induction of apoptotic cell death [11]. ROS generation by high glucose has been shown to trigger mitochondrial damage [22] and release cytochrome c [23]. Mitochondrial cytochrome c release is recognized to be a key event in the activation of caspase-3, which plays a pivotal role in the execution of apoptosis [24]. Recently, Epo has been identified as an important mediator of the adaptive responses to metabolilc stress. It also has been shown by several research groups to possess potent neuroprotective properties in experimental cerebral, retinal and motor neuron ischemia [15,16,25,26]. Since Epo receptors are expressed on vascular endothelial cells as well as erythroid cells and neurons, Epo is implicated to possess vascular-protective effect [27]. Concerning the mechanism of Epo’s effect, several signal transduction pathways may be involved. Akt, which is a downstream of PI-3 kinase, is an important factor of cell survival, because Akt activation ultimately results in uregulation of the bcl family of anti-apoptotic genes. Based on the conception, we examined the contribution of PI-3 kinase pathway to the anti-apoptotic effect of Epo. In this study, we have presented that Epo phosphorylates Akt in dose-dependent manner and plays a role against endothelial cell apoptosis induced by high glucose. Blockade of PI-3 kinase by wormannin and LY294002 confirm the contribution of Epo to the PI-3 kinase pathway. In conclusion, we revealed that Epo can inhibit an apoptotis of endothelial cells induced by high glucose. It is also shown that Epo ameliorates the apoptotic reaction through PI-3 kinase pathway. These results suggest that reduced production of EPO in patients with end-stage of nephropathy may accelerate diabetic angiopathy and that replacing therapy with EPO might inhibit endothelial cell apoptosis and prevent diabetic angiopathy.

Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (No. 11671126) from the Ministry of Education, Science and Culture, Japan. References [1] B.A. O’Brien, B.V. Harmon, D.P. Cameron, D.J. Allan, Apoptosis is the mode of beta-cell death responsible for the development of IDDM in the nonobese diabetic (NOD) mouse, Diabetes 46 (1997) 750–757. [2] R. Lupi, F. Dotta, L. Marselli, et al., Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets: evidence that beta-cell death is caspase mediated, partially dependent on ceramide pathway, and Bcl-2 regulated, Diabetes 51 (2002) 1437–1442. [3] S. Gorogawa, Y. Kajimoto, Y. Umayahara, et al., Probucol preserves pancreatic beta-cell function through reduction of oxidative stress in type 2 diabetes, Diab. Res. Clin. Pract. 57 (2002) 1–10. [4] D.R. Laybutt, G.C. Weir, H. Kaneto, et al., Overexpression of c-Myc in beta-cells of transgenic mice causes proliferation and apoptosis, downregulation of insulin gene expression, and diabetes, Diabetes 51 (2002) 1793–1804. [5] A.Y. Savinov, A. Tcherepanov, E.A. Green, R.A. Flavell, A.V. Chervonsky, Contribution of Fas to diabetes development, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 628–632. [6] M. Mizutani, T.S. Kern, M. Lorenzi, Accelerated death of retinal microvascular cells in human and experimental diabetic retinopathy, J. Clin. Invest. 97 (1996) 2883–2890. [7] F. Podesta, G. Romeo, W.H. Liu, et al., Bax is increased in the retina of diabetic subjects and is associated with pericyte apoptosis in vivo and in vitro, Am. J. Pathol. 156 (2000) 1025–1032. [8] S. Mohr, X. Xi, J. Tang, T.S. Kern, Caspase activation in retinas of diabetic and galactosemic mice and diabetic patients, Diabetes 51 (2002) 1172–1179. [9] S. Yamagishi, Y. Inagaki, T. Okamoto, et al., Advanced glycation end product-induced apoptosis and overexpression of vascular endothelial growth factor and monocyte chemoattractant protein-1 in human-cultured mesangial cells, J. Biol. Chem. 277 (2002) 20309–20315. [10] S.M. Baumgartner-Parzer, L. Wagner, M. Pettermann, J. Grillari, A. Gessl, W. Waldhausl, High-glucose-triggered apoptosis in cultured endothelial cells, Diabetes 44 (1995) 1323–1327. [11] F.M. Ho, S.H. Liu, C.S. Liau, P.J. Huang, S.Y. Lin-Shiau, High glucose-induced apoptosis in human endothelial cells is mediated by sequential activations of c-Jun NH(2)-terminal kinase and caspase-3, Circulation 101 (2000) 2618–2624. [12] C. Min, E. Kang, S.H. Yu, S.H. Shinn, Y.S. Kim, Advanced glycation end products induce apoptosis and procoagulant activity in cultured human umbilical vein endothelial cells, Diab. Res. Clin. Pract. 46 (1999) 197–202.

N. Sekiguchi et al. / Diabetes Research and Clinical Practice 66S (2004) S103–S107 [13] M. Artwohl, W.F. Graier, M. Roden, et al., Diabetic LDL triggers apoptosis in vascular endothelial cells, Diabetes 52 (2003) 1240–1247. [14] A.M. Schmeichel, J.D. Schmelzer, P.A. Low, Oxidative injury and apoptosis of dorsal root ganglion neurons in chronic experimental diabetic neuropathy, Diabetes 52 (2003) 165–171. [15] M. Celik, N. Gokmen, S. Erbayraktar, Erythropoietin prevents motor neuron apoptosis and neurologic disability in experimental spinal cord ischemic injury, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 2258–2263. [16] A.L. Siren, M. Fratelli, M. Brines, et al., Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 4044–4049. [17] E. Bossy-Wetzel, D.D. Newmeyer, D.R. Green, Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization, EMBO J. 17 (1998) 37–49. [18] J. Xu, C.H. Yeh, S. Chen, et al., Involvement of de novo ceramide biosynthesis in tumor necrosis factor-alpha/ cycloheximide-induced cerebral endothelial cell death, J. Biol. Chem. 273 (1998) 16521–16526. [19] N.A. Thornberry, Y. Lazebnik, Caspases: enemies within, Science 281 (1998) 1312–1316. [20] X.M. Sun, M. MacFarlane, J. Zhuang, B.B. Wolf, D.R. Green, G.M. Cohen, Distinct caspase cascades are initiated

[21]

[22]

[23] [24]

[25]

[26]

[27]

S107

in receptor-mediated and chemical-induced apoptosis, J. Biol. Chem. 274 (1999) 5053–5060. X.L. Du, G.Z. Sui, K. Stockklauser-Farber, et al., Introduction of apoptosis by high proinsulin and glucose in cultured human umbilical vein endothelial cells is mediated by reactive oxygen species, Diabetologia 41 (1998) 249– 256. J.W. Baynes, S.R. Thorpe, Role of oxidative stress in diabetic complications: a new perspective on an old paradigm, Diabetes 48 (1999) 1–9. S. Roy, Caspases at the heart of the apoptotic cell death pathway, Chem. Res. Toxicol. 13 (2000) 961–962. W. Wu, W.L. Lee, Y.Y. Wu, et al., Expression of constitutively active phosphatidylinositol 3-kinase inhibits activation of caspase-3 and apoptosis of cardiac muscle cells, J. Biol. Chem. 275 (2000) 40113–40119. M.L. Brines, P. Ghezzi, S. Keenan, et al., Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 10526– 10531. C. Grimm, A. Wenzel, M. Groszer, et al., HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration, Nat. Med. 8 (2002) 718– 724. A. Anagnostou, Z. Liu, M. Steiner, et al., Erythropoietin receptor mRNA expression in human endothelial cells, Proc. Natl. Acad. Sci. U.S.A. 91 (1994) 3974–3978.