Evaluation of Energy Balance on Human Telomerase Reverse Transcriptase (hTERT) Alternative Splicing by Semi-quantitative RT-PCR in Human Umbilical Vein Endothelial Cells


1 Heart Failure Research Center, Cardiovascular Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan, Iran

2 Department of Cardiology, Isfahan University of Medical Sciences, Isfahan, Iran

3 Department of Genetics and Molecular Biology, Isfahan University of Medical Sciences, Isfahan, Iran

4 Applied Physiology Research Center, Isfahan Cardiovascular Research Institute, Department of Physiology, Isfahan University of Medical Sciences, Isfahan, Iran


Background: Decreased high-energy phosphate level is involved in endothelial cell injury and dysfunction. Reduced telomerase activity in endothelial cells in parallel with reduced energy levels might be due to altered direction of alternative splicing machine as a complication of depleted energy during the process of atherosclerosis. Materials and Methods: Isolated human umbilical vein endothelial cells (HUVECs) were treated for 24 hours by oligomycine (OM) and 2-deoxy glucose (2-DG). After 24 hours, the effect of energy depletion on telomerase splicing pattern was evaluated using RT-PCR. Indeed, in both treated and untargeted cells, nitric oxide (NO) and von Willebrand factor (vWF) were measured. Results: ATP was depleted in treated cells by 43.9% compared with control group. We observed a slight decrease in NO levels (P = 0.09) and vWF (P = 0.395) in the setting of 49.36% ATP depletion. In both groups, no telomerase gene expression was seen. Telomerase and housekeeping gene expression were found in positive control group (colon cancer tissue) and sample tissue. Conclusions: The absence of telomerase gene expression in HUVECs might be due to the mortality of these cells or the low level of telomerase gene expression in these cells under normal circumstances.


Kilian A, Bowtell DD, Abud HE, Hime GR, Venter DJ, Keese PK, et al. Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types. Hum Mol Genet 1997;6:2011-9.  Back to cited text no. 1
Derradji H, Bekaert S, Van Oostveldt P, Baatout S. Comparison of different protocols for telomere length estimation by combination of quantitative fluorescence in situ hybridization (Q-FISH) and flow cytometry in human cancer cell lines. Anticancer Res 2005;25:1039-50.  Back to cited text no. 2
Pandita TK, Roti Roti JL. Role of telomerase in radiocurability (review). Oncol Rep 2003;10:263-70.  Back to cited text no. 3
Lantuéjoul S, Salon C, Soria JC, Brambilla E. Telomerase expression in lung preneoplasia and neoplasia. Int J Cancer 2007;120:1835-41.  Back to cited text no. 4
Liu WJ, Zhang YW, Zhang ZX, Ding J. Alternative splicing of human telomerase reverse transcriptase may not be involved in telomerase regulation during all-trans-retinoic acid-induced HL-60 cell differentiation. J Pharmacol Sci 2004;96:106-14.  Back to cited text no. 5
Ulaner GA, Hu JF, Vu TH, Giudice LC, Hoffman AR. Tissue-specific alternate splicing of human telomerase reverse transcriptase (hTERT) influences telomere lengths during human development. Int J Cancer 2001;91:644-9.  Back to cited text no. 6
Pu DR, Liu L. HDL slowing down endothelial progenitor cells senescence: A novel anti-atherogenic property of HDL. Med Hypotheses 2008;70:338-42.  Back to cited text no. 7
Minamino T, Miyauchi H, Yoshida T, Ishida Y, Yoshida H, Komuro I. Endothelial cell senescence in human atherosclerosis: Role of telomere in endothelial dysfunction. Circulation 2002;105:1541-4.  Back to cited text no. 8
Minamino T, Komuro I. Role of telomere in endothelial dysfunction in atherosclerosis. Curr Opin Lipidol 2002;13:537-43.  Back to cited text no. 9
Jian W, Arora JS, Oe T, Shuvaev VV, Blair IA. Induction of endothelial cell apoptosis by lipid hydroperoxide-derived bifunctional electrophiles. Free Radic Biol Med 2005;39:1162-76.  Back to cited text no. 10
Beldi G, Enjyoji K, Wu Y, Miller L, Banz Y, Sun X, et al. The role of purinergic signaling in the liver and in transplantation: Effects of extracellular nucleotides on hepatic graft vascular injury, rejection and metabolism. Front Biosci 2008;13:2588-603.  Back to cited text no. 11
Mathews MT, Berk BC. PARP-1 inhibition prevents oxidative and nitrosative stress-induced endothelial cell death via transactivation of the VEGF receptor 2. Arterioscler Thromb Vasc Biol 2008;28:711-7.  Back to cited text no. 12
Carlucci F, Marinello E, Rosi F, Floccari F, Gerunda G, Neri D, et al. Nitric oxide generation is associated with an unbalance of protein tyrosine phosphatases during liver transplantation. Biomed Pharmacother 2007;61:216-21.  Back to cited text no. 13
Hurst RD, Azam S, Hurst A, Clark JB. Nitric-oxide-induced inhibition of glyceraldehyde-3-phosphate dehydrogenase may mediate reduced endothelial cell monolayer integrity in an in vitro model blood-brain barrier. Brain Res 2001;894:181-8.  Back to cited text no. 14
Hermans MP. Diabetes and the endothelium. Acta Clin Belg 2007;62:97-101.  Back to cited text no. 15
Agulló L, Garcia-Dorado D, Escalona N, Inserte J, Ruiz-Meana M, Barrabés JA, et al. Hypoxia and acidosis impair cGMP synthesis in microvascular coronary endothelial cells. Am J Physiol Heart Circ Physiol 2002;283:H917-25.  Back to cited text no. 16
de la Torre JC, Stefano GB. Evidence that Alzheimer's disease is a microvascular disorder: The role of constitutive nitric oxide. Brain Res Brain Res Rev 2000;34:119-36.  Back to cited text no. 17
Ciapaite J, van Bezu J, van Eikenhorst G, Bakker SJ, Teerlink T, Diamant M, et al. Palmitate and oleate have distinct effects on the inflammatory phenotype of human endothelial cells. Biochim Biophys Acta 2007;1771:147-54.  Back to cited text no. 18
Razmara A, Sunday L, Stirone C, Wang XB, Krause DN, Duckles SP, et al. Mitochondrial effects of estrogen are mediated by estrogen receptor alpha in brain endothelial cells. J Pharmacol Exp Ther 2008;325:782-90.  Back to cited text no. 19
Krainer AR, Maniatis T, Ruskin B, Green MR. Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell 1984;36:993-1005.  Back to cited text no. 20
Hartl DL, Elizabeth W. Genetics: Analysis of Genes and Genomes. Sudbury, Massachusetts: Jones and Bartlett Publishers [My paper] 2005; Jan 1, 2005.  Back to cited text no. 21
Ulaner GA, Hu JF, Vu TH, Giudice LC, Hoffman AR. Tissue-specific alternate splicing of human telomerase reverse transcriptase (hTERT) influences telomere lengths during human development. Int J Cancer 2001;91:644-9.  Back to cited text no. 22
Behjati M, Hashemi M, Kazemi M, Salehi M. Nitric oxide level and von Willebrand factor (vWF) secretion are not candidate markers of endothelial cell dysfunction in adenosine triphosphate (ATP) depleted endothelial cells. Afr J Biotechnol 2011;10:8871-6.  Back to cited text no. 23
Noguchi H, Kobayashi N, Westerman KA, Sakaguchi M, Okitsu T, Totsugawa T, et al. Controlled expansion of human endothelial cell populations by Cre-loxP -based reversible immortalization. Hum Gene Ther 2002;13:321-34.  Back to cited text no. 24
Tentori L, Vergati M, Muzi A, Levati L, Ruffini F, Forini O, et al. Generation of an immortalized human endothelial cell line as a model of neovascular proliferating endothelial cells to assess chemosensitivity to anticancer drugs. Int J Oncol 2005;27:525-35.  Back to cited text no. 25
Falchetti ML, Mongiardi MP, Fiorenzo P, Petrucci G, Pierconti F, D'Agnano I, et al. Inhibition of telomerase in the endothelial cells disrupts tumor angiogenesis in glioblastoma xenografts. Int J Cancer 2008;122:1236-42.  Back to cited text no. 26
Chang H, Rha SY, Jeung HC, Park KH, Kim TS, Kim YB, et al. Telomerase- and angiogenesis -related gene responses to irradiation in human umbilical vein endothelial cells. Int J Mol Med 2013;31:1202-8.  Back to cited text no. 27
Ling S, Zhou L, Li H, Dai A, Liu JP, Komesaroff PA, et al. Effects of 17 beta-estradiol on growth and apoptosis in human vascular endothelial cells: Influence of mechanical strain and tumor necrosis factor-alpha. Steroids 2006;71:799-808.  Back to cited text no. 28
Vasa M, Breitschopf K, Zeiher AM, Dimmeler S. Nitric oxide activates telomerase and delays endothelial cell senescence. Circ Res 2000;87:540-2.  Back to cited text no. 29
Mortuza R, Chen S, Feng B, Sen S, Chakrabarti S. High glucose induced alteration of SIRTs in endothelial cells causes rapid aging in a p300 and FOXO regulated pathway. PLoS One 2013;8:e54514.  Back to cited text no. 30
Takano H, Murasawa S, Asahara T. Functional and gene expression analysis of hTERT over expressed endothelial cells. Biologics 2008;2:547-54.  Back to cited text no. 31
Ingram DA, Mead LE, Moore DB, Woodard W, Fenoglio A, Yoder MC. Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells. Blood 2005;105:2783-6.  Back to cited text no. 32