Effects of Empagliflozin on Intermittent Hypoxia-Induced TRAF3IP2-Dependent Human Aortic Smooth Muscle Cell Proliferation

Main Article Content

Ryan Dashek Yusuke Higashi Nitin A. Das Jacob J. Russell Luis A. Martinez-Lemus R. Scott Rector Bysani Chandrasekar


Aims: Chronic intermittent hypoxia (IH), a characteristic feature of obstructive sleep apnea (OSA), contributes to cardiovascular diseases, including atherosclerosis, potentially through persistent oxidative stress and inflammation. TRAF3IP2 (TRAF3 Interacting Protein 2) is an oxidative stress-responsive proinflammatory adapter molecule and plays a causal role in a preclinical model of atherosclerosis. Since SGLT2 (Sodium/Glucose Cotransporter 2) inhibitors have shown protective effects in CVD by inhibiting oxidative stress and inflammation, we hypothesized that IH promotes the crosstalk between oxidative stress and TRAF3IP2, resulting in IL-6-dependent human aortic smooth muscle cell (SMC) proliferation, and that these effects are inhibited by the SGLT2 inhibitor empagliflozin.

Materials and methods: Primary human aortic SMC were exposed to various cycles of IH. Normoxia served as a control. To understand the molecular mechanisms underlying IH-induced nitroxidative stress, TRAF3IP2 and IL-6 induction, and SMC proliferation and those targeted by empagliflozin were determined by treating SMC with various pharmacological inhibitors and viral vectors.

Results: IH upregulated TRAF3IP2 expression, TRAF3IP2-dependent superoxide, hydrogen peroxide and nitric oxide generation, NF-kB and HIF-1a activation, IL-6 induction, and SMC proliferation. Exposure to IL-6 by itself induced SMC proliferation in part via TRAF3IP2, IL-6R, gp130, JAK, and STAT3. Further, SMC express SGLT2 at basal conditions, and is upregulated by both IH and IL-6. Importantly, empagliflozin inhibited IH-induced TRAF3IP2 upregulation, reactive oxygen and nitrogen species generation, TRAF3IP2-dependent HIF-1a and NF-kB activation, IL-6 induction, and IL-6-dependent JAK-STAT3-mediated SMC proliferation. Moreover, empagliflozin inhibited IL-6-induced STAT3-dependent SMC proliferation.

Conclusions: These results suggest the therapeutic potential of empagliflozin in IH and inflammatory vascular proliferative diseases associated with OSA.


Article Details

How to Cite
DASHEK, Ryan et al. Effects of Empagliflozin on Intermittent Hypoxia-Induced TRAF3IP2-Dependent Human Aortic Smooth Muscle Cell Proliferation. Medical Research Archives, [S.l.], v. 10, n. 10, oct. 2022. ISSN 2375-1924. Available at: <https://esmed.org/MRA/mra/article/view/3237>. Date accessed: 06 dec. 2022. doi: https://doi.org/10.18103/mra.v10i10.3237.
Research Articles


1. Peppard PE, Young T, Barnet JH, Palta M, Hagen EW, Hla KM. Increased prevalence of sleep-disordered breathing in adults. Am J Epidemiol. May 1 2013;177(9):1006-14. doi:10.1093/aje/kws342
2. Dewan NA, Nieto FJ, Somers VK. Intermittent hypoxemia and OSA: implications for comorbidities. Chest. Jan 2015;147(1):266-274. doi:10.1378/chest.14-0500
3. Kyotani Y, Ota H, Itaya-Hironaka A, et al. Intermittent hypoxia induces the proliferation of rat vascular smooth muscle cell with the increases in epidermal growth factor family and erbB2 receptor. Exp Cell Res. Nov 15 2013;319(19):3042-50. doi:10.1016/j.yexcr.2013.08.014
4. Arnaud C, Beguin PC, Lantuejoul S, et al. The inflammatory preatherosclerotic remodeling induced by intermittent hypoxia is attenuated by RANTES/CCL5 inhibition. Am J Respir Crit Care Med. Sep 15 2011;184(6):724-31. doi:10.1164/rccm.201012-2033OC
5. Leonardi A, Chariot A, Claudio E, Cunningham K, Siebenlist U. CIKS, a connection to Ikappa B kinase and stress-activated protein kinase. Proc Natl Acad Sci U S A. Sep 12 2000;97(19):10494-9. doi:10.1073/pnas.190245697
6. Li X, Commane M, Nie H, et al. Act1, an NF-kappa B-activating protein. Proc Natl Acad Sci U S A. Sep 12 2000;97(19):10489-93. doi:10.1073/pnas.160265197
7. Das NA, Carpenter AJ, Yoshida T, et al. TRAF3IP2 mediates TWEAK/TWEAKR-induced pro-fibrotic responses in cultured cardiac fibroblasts and the heart. J Mol Cell Cardiol. Aug 2018;121:107-123. doi:10.1016/j.yjmcc.2018.07.003
8. Valente AJ, Irimpen AM, Siebenlist U, Chandrasekar B. OxLDL induces endothelial dysfunction and death via TRAF3IP2: inhibition by HDL3 and AMPK activators. Free Radic Biol Med. May 2014;70:117-28. doi:10.1016/j.freeradbiomed.2014.02.014
9. Chen S, Crother TR, Arditi M. Emerging role of IL-17 in atherosclerosis. J Innate Immun. 2010;2(4):325-33. doi:10.1159/000314626
10. Mummidi S, Das NA, Carpenter AJ, et al. RECK suppresses interleukin-17/TRAF3IP2-mediated MMP-13 activation and human aortic smooth muscle cell migration and proliferation. J Cell Physiol. Dec 2019;234(12):22242-22259. doi:10.1002/jcp.28792
11. Sakamuri S, Higashi Y, Sukhanov S, et al. TRAF3IP2 mediates atherosclerotic plaque development and vulnerability in ApoE(-/-) mice. Atherosclerosis. Sep 2016;252:153-160. doi:10.1016/j.atherosclerosis.2016.05.029
12. Hasan FM, Alsahli M, Gerich JE. SGLT2 inhibitors in the treatment of type 2 diabetes. Diabetes Res Clin Pract. Jun 2014;104(3):297-322. doi:10.1016/j.diabres.2014.02.014
13. Packer M, Anker SD, Butler J, et al. Cardiovascular and Renal Outcomes with Empagliflozin in Heart Failure. N Engl J Med. Oct 8 2020;383(15):1413-1424. doi:10.1056/NEJMoa2022190
14. Anker SD, Butler J, Filippatos G, et al. Empagliflozin in Heart Failure with a Preserved Ejection Fraction. N Engl J Med. Oct 14 2021;385(16):1451-1461. doi:10.1056/NEJMoa2107038
15. Das NA, Carpenter AJ, Belenchia A, et al. Empagliflozin reduces high glucose-induced oxidative stress and miR-21-dependent TRAF3IP2 induction and RECK suppression, and inhibits human renal proximal tubular epithelial cell migration and epithelial-to-mesenchymal transition. Cell Signal. Apr 2020;68:109506. doi:10.1016/j.cellsig.2019.109506
16. Aroor AR, Das NA, Carpenter AJ, et al. Glycemic control by the SGLT2 inhibitor empagliflozin decreases aortic stiffness, renal resistivity index and kidney injury. Cardiovasc Diabetol. Jul 30 2018;17(1):108. doi:10.1186/s12933-018-0750-8
17. Palombo C, Kozakova M. Arterial stiffness, atherosclerosis and cardiovascular risk: Pathophysiologic mechanisms and emerging clinical indications. Vascul Pharmacol. Feb 2016;77:1-7. doi:10.1016/j.vph.2015.11.083
18. Behnammanesh G, Durante GL, Khanna YP, Peyton KJ, Durante W. Canagliflozin inhibits vascular smooth muscle cell proliferation and migration: Role of heme oxygenase-1. Redox Biol. May 2020;32:101527. doi:10.1016/j.redox.2020.101527
19. Sukhanov S, Higashi Y, Yoshida T, et al. The SGLT2 inhibitor Empagliflozin attenuates interleukin-17A-induced human aortic smooth muscle cell proliferation and migration by targeting TRAF3IP2/ROS/NLRP3/Caspase-1-dependent IL-1beta and IL-18 secretion. Cell Signal. Jan 2021;77:109825. doi:10.1016/j.cellsig.2020.109825
20. Xu S, Grande F, Garofalo A, Neamati N. Discovery of a novel orally active small-molecule gp130 inhibitor for the treatment of ovarian cancer. Mol Cancer Ther. Jun 2013;12(6):937-49. doi:10.1158/1535-7163.MCT-12-1082
21. Nakane M, Klinghofer V, Kuk JE, et al. Novel potent and selective inhibitors of inducible nitric oxide synthase. Mol Pharmacol. Apr 1995;47(4):831-4.
22. Somanna NK, Valente AJ, Krenz M, Fay WP, Delafontaine P, Chandrasekar B. The Nox1/4 Dual Inhibitor GKT137831 or Nox4 Knockdown Inhibits Angiotensin-II-Induced Adult Mouse Cardiac Fibroblast Proliferation and Migration. AT1 Physically Associates With Nox4. J Cell Physiol. May 2016;231(5):1130-41. doi:10.1002/jcp.25210
23. Scheen AJ. Pharmacokinetic and pharmacodynamic profile of empagliflozin, a sodium glucose co-transporter 2 inhibitor. Clin Pharmacokinet. Mar 2014;53(3):213-225. doi:10.1007/s40262-013-0126-x
24. Wang N, Shi XF, Khan SA, et al. Hypoxia-inducible factor-1 mediates pancreatic beta-cell dysfunction by intermittent hypoxia. Am J Physiol Cell Physiol. Nov 1 2020;319(5):C922-C932. doi:10.1152/ajpcell.00309.2020
25. Higashi Y, Mummidi S, Sukhanov S, et al. Minocycline inhibits PDGF-BB-induced human aortic smooth muscle cell proliferation and migration by reversing miR-221- and -222-mediated RECK suppression. Cell Signal. May 2019;57:10-20. doi:10.1016/j.cellsig.2019.01.014
26. Hosoi T, Okuma Y, Kawagishi T, Qi X, Matsuda T, Nomura Y. Bacterial endotoxin induces STAT3 activation in the mouse brain. Brain Res. Oct 8 2004;1023(1):48-53. doi:10.1016/j.brainres.2004.06.076
27. Lavie L. Sleep-disordered breathing and cerebrovascular disease: a mechanistic approach. Neurol Clin. Nov 2005;23(4):1059-75. doi:10.1016/j.ncl.2005.05.005
28. Garvey JF, Taylor CT, McNicholas WT. Cardiovascular disease in obstructive sleep apnoea syndrome: the role of intermittent hypoxia and inflammation. Eur Respir J. May 2009;33(5):1195-205. doi:10.1183/09031936.00111208
29. Erikson JM, Valente AJ, Mummidi S, et al. Targeting TRAF3IP2 by Genetic and Interventional Approaches Inhibits Ischemia/Reperfusion-induced Myocardial Injury and Adverse Remodeling. J Biol Chem. Feb 10 2017;292(6):2345-2358. doi:10.1074/jbc.M116.764522
30. Valente AJ, Clark RA, Siddesha JM, Siebenlist U, Chandrasekar B. CIKS (Act1 or TRAF3IP2) mediates Angiotensin-II-induced Interleukin-18 expression, and Nox2-dependent cardiomyocyte hypertrophy. J Mol Cell Cardiol. Jul 2012;53(1):113-24. doi:10.1016/j.yjmcc.2012.04.009
31. Sakamuri SS, Valente AJ, Siddesha JM, et al. TRAF3IP2 mediates aldosterone/salt-induced cardiac hypertrophy and fibrosis. Mol Cell Endocrinol. Jul 5 2016;429:84-92. doi:10.1016/j.mce.2016.03.038
32. Schulz R, Mahmoudi S, Hattar K, et al. Enhanced release of superoxide from polymorphonuclear neutrophils in obstructive sleep apnea. Impact of continuous positive airway pressure therapy. Am J Respir Crit Care Med. Aug 2000;162(2 Pt 1):566-70. doi:10.1164/ajrccm.162.2.9908091
33. Del Ben M, Fabiani M, Loffredo L, et al. Oxidative stress mediated arterial dysfunction in patients with obstructive sleep apnoea and the effect of continuous positive airway pressure treatment. BMC Pulm Med. Jul 23 2012;12:36. doi:10.1186/1471-2466-12-36
34. Htoo AK, Greenberg H, Tongia S, et al. Activation of nuclear factor kappaB in obstructive sleep apnea: a pathway leading to systemic inflammation. Sleep Breath. Mar 2006;10(1):43-50. doi:10.1007/s11325-005-0046-6
35. Dyugovskaya L, Polyakov A, Cohen-Kaplan V, Lavie P, Lavie L. Bax/Mcl-1 balance affects neutrophil survival in intermittent hypoxia and obstructive sleep apnea: effects of p38MAPK and ERK1/2 signaling. J Transl Med. Oct 22 2012;10:211. doi:10.1186/1479-5876-10-211
36. Minet E, Michel G, Mottet D, Raes M, Michiels C. Transduction pathways involved in Hypoxia-Inducible Factor-1 phosphorylation and activation. Free Radic Biol Med. Oct 1 2001;31(7):847-55. doi:10.1016/s0891-5849(01)00657-8
37. van Uden P, Kenneth NS, Rocha S. Regulation of hypoxia-inducible factor-1alpha by NF-kappaB. Biochem J. Jun 15 2008;412(3):477-84. doi:10.1042/BJ20080476
38. Scortegagna M, Cataisson C, Martin RJ, et al. HIF-1alpha regulates epithelial inflammation by cell autonomous NFkappaB activation and paracrine stromal remodeling. Blood. Apr 1 2008;111(7):3343-54. doi:10.1182/blood-2007-10-115758
39. Bandarra D, Biddlestone J, Mudie S, Muller HA, Rocha S. HIF-1alpha restricts NF-kappaB-dependent gene expression to control innate immunity signals. Dis Model Mech. Feb 2015;8(2):169-81. doi:10.1242/dmm.017285
40. Imani MM, Sadeghi M, Khazaie H, Emami M, Sadeghi Bahmani D, Brand S. Evaluation of Serum and Plasma Interleukin-6 Levels in Obstructive Sleep Apnea Syndrome: A Meta-Analysis and Meta-Regression. Front Immunol. 2020;11:1343. doi:10.3389/fimmu.2020.01343
41. Woods A, Brull DJ, Humphries SE, Montgomery HE. Genetics of inflammation and risk of coronary artery disease: the central role of interleukin-6. Eur Heart J. Oct 2000;21(19):1574-83. doi:10.1053/euhj.1999.2207
42. Fu M, Yu J, Chen Z, et al. Epoxyeicosatrienoic acids improve glucose homeostasis by preventing NF-kappaB-mediated transcription of SGLT2 in renal tubular epithelial cells. Mol Cell Endocrinol. Mar 1 2021;523:111149. doi:10.1016/j.mce.2020.111149
43. Neeland IJ, Eliasson B, Kasai T, et al. The Impact of Empagliflozin on Obstructive Sleep Apnea and Cardiovascular and Renal Outcomes: An Exploratory Analysis of the EMPA-REG OUTCOME Trial. Diabetes Care. Dec 2020;43(12):3007-3015. doi:10.2337/dc20-1096