Korean Circ J.  2020 May;50(5):443-457. 10.4070/kcj.2019.0296.

Anti-Inflammatory Effect for Atherosclerosis Progression by Sodium-Glucose Cotransporter 2 (SGLT-2) Inhibitor in a Normoglycemic Rabbit Model

Affiliations
  • 1Yonsei Cardiovascular Research Institute, Yonsei University College of Medicine, Seoul, Korea. KJS1218@yuhs.ac
  • 2Cardiology Division, Severance Cardiovascular Hospital, Yonsei University College of Medicine, Seoul, Korea.
  • 3Graduate Program in Science for Aging, Yonsei University, Seoul, Korea.
  • 4Cardiovascular Product Evaluation Center, Yonsei University College of Medicine, Seoul, Korea.
  • 5Division of Cardiology, Yongin Severance Hospital, Yonsei University College of Medicine, Yongin, Korea.
  • 6Department of Cardiology, Ewha Womans University Seoul Hospital, Seoul, Korea.
  • 7Division of Cardiology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
  • 8Division of Endocrinology and Metabolism, Department of Internal Medicine, Severance Hospital, Yonsei University College of Medicine, Seoul, Korea.

Abstract

BACKGROUND AND OBJECTIVES
We sought to investigate an anti-atherosclerotic and anti-inflammatory effect of sodium-glucose cotransporter-2 (SGLT-2) inhibitors in normoglycemic atherosclerotic rabbit model.
METHODS
Male New Zealand white rabbits (n=26) were fed with a 1% high-cholesterol diet for 7 weeks followed by normal diet for 2 weeks. After balloon catheter injury, the rabbits were administered with the Dapagliflozin (1mg/kg/day) or control-medium for 8 weeks (n=13 for each group). All lesions were assessed with angiography, optical coherence tomography (OCT), and histological assessment.
RESULTS
Atheroma burden (38.51±3.16% vs. 21.91±1.22%, p<0.01) and lipid accumulation (18.90±3.63% vs. 10.20±2.03%, p=0.047) was significantly decreased by SGLT-2 inhibitor treatment. The SGLT-2 inhibitor group showed lower macrophage infiltration (20.23±1.89% vs. 12.72±1.95%, p=0.01) as well as tumor necrosis factor (TNF)-α expression (31.17±4.40% vs. 19.47±2.10%, p=0.025). Relative area of inducible nitric oxide synthase+ macrophages was tended to be lower in the SGLT-2 inhibitor-treated group (1.00±0.16% vs. 0.71±0.10%, p=0.13), while relative proportion of Arg1⁺ macrophage was markedly increased (1.00±0.27% vs. 2.43±0.64%, p=0.04). As a result, progression of atherosclerosis was markedly attenuated in SGLT-2 inhibitor treated group (OCT area stenosis, 32.13±1.20% vs. 22.77±0.88%, p<0.01). Mechanistically, SGLT-2 treatment mitigated the inflammatory responses in macrophage. Especially, Toll-like receptor 4/nuclear factor-kappa B signaling pathway, and their downstream effectors such as interleukin-6 and TNF-α were markedly suppressed by SGLT-2 inhibitor treatment.
CONCLUSIONS
These results together suggest that SGLT-2 inhibitor exerts an anti-atherosclerotic effect through favorable modulation of inflammatory response as well as macrophage characteristics in non-diabetic situation.

Keyword

Atherosclerosis; Sodium-glucose transporter-2; Sodium-glucose transporter 2 inhibitors; Macrophages

MeSH Terms

Angiography
Atherosclerosis*
Catheters
Constriction, Pathologic
Diet
Humans
Interleukin-6
Macrophages
Male
Nitric Oxide
Plaque, Atherosclerotic
Rabbits
Toll-Like Receptors
Tomography, Optical Coherence
Tumor Necrosis Factor-alpha
Interleukin-6
Nitric Oxide
Toll-Like Receptors
Tumor Necrosis Factor-alpha

Figure

  • Figure 1 Schematic design of the study protocol. After 1 week of a high-cholesterol diet, balloon injury was induced in the abdominal aorta. Rabbits then received a SGLT-2i, depending on their group assignment, for 8 weeks. Blood was collected baseline and before sacrifice at the end. OCT was assessed at the end of the study, and then the aorta was harvested.OCT = optical coherence tomography; SGLT-2i = sodium-glucose cotransporter-2 inhibitor.

  • Figure 2 Imaging analyses for the progression of atherosclerosis. (A) Angiography compared diameter stenosis of each group at baseline and follow up. (B) OCT images showing area stenosis for each group. The red arrows point to the lipid. (C) Tissues were stained with H&E, ORO, trichrome and pentachrome. (D) Atheromatous plaque for vessels in each group. (E) Lipid accumulation of plaques analyzed by ORO staining. Scale bars represent 100 µm. Data are mean±standard error of the mean.Dapa = dapagliflozin; H&E = hematoxylin and eosin; OCT = optical coherence tomography; ORO = Oil Red O.*p<0.05 vs. Control group.

  • Figure 3 IHC staining of macrophage and inflammation markers. (A) Tissues immunologically stained of RAM11 positive area. (B) Tissues immunologically stained of TNF-α positive area. (C) Tissues immunologically stained of IL-1β positive area. (D) Tissues immunologically stained of IL-6 positive area. Scale bars represent 100 µm. Data are mean±standard error of the mean.Dapa = dapagliflozin; IHC = immunohistochemical; IL = interleukin; TNF = tumor necrosis factor.*p<0.05 vs. Control group.

  • Figure 4 Macrophage polarization assessed by confocal immunofluorescence microscopy. (A) Confocal immunofluorescence microscopy showing CD68 (green), iNOS (red), and Arg-1 (white) localization in the same vessel. Blue represents 4′,6-diamidino-2-phenylindole staining of nuclei. (B) Relative area of CD68–iNOS staining in each group. (C) Relative area of CD68–Arg-1 staining in each group. (D) Arg-1/iNOS ratio in each group. n=10 (each slide of 3 sections). Relative area measurements were determined using a Zeiss LSM 700. Scale bars represent 100 μm. Data are mean±standard error of the mean.Arg-1 = arginase-1; Dapa = dapagliflozin; DAPI = 4′,6-diamidino-2-phenylindole; iNOS = inducible nitric oxide synthase.*p<0.05 vs. Control group.

  • Figure 5 Real-time RT-PCR and Western blot analysis of aorta artery. (A-E) RT-PCR expression of IL-1β, IL-6, TNF-α, iNOS and Arg-1. Comparisons of relative mRNA expression, normalized to expression of GAPDH as the housekeeping gene. (F) mRNA expression of Arg-1/iNOS ratio. (G-J) Western blot expression of TNF-α, NF-κB, iNOS, and Arg-1. (K) Protein expression of Arg-1/iNOS ratio. Representative data showing mRNA and protein expression, normalized to expression of GAPDH as the housekeeping gene. Data in the bar graphs are quantified ratios of each signal relative to the signal for GAPDH, presented as fold increases. Data are mean±standard error of the mean.Arg-1 = arginase-1; Dapa = dapagliflozin; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; IL = interleukin; iNOS = inducible nitric oxide synthase; NF-κB = nuclear factor-kappa B; RT-PCR = reverse transcription-polymerase chain reaction; TNF = tumor necrosis factor.*p<0.05 vs. Control group.

  • Figure 6 RT-PCR and Western blot analysis of RAW264.7. (A-D) RT-PCR expression of TLR4, NF-κB, IL-6 and TNF-α. Comparisons of relative mRNA expression, normalized to expression of β-actin as the housekeeping gene. (E) Western blot expression of TLR4, NF-κB, IL-6 and TNF-α. (F-I) Comparisons of relative protein expression, normalized to expression of GAPDH as the housekeeping gene. Data in the bar graphs are quantified ratios of the each signal relative to the signal for housekeeping gene, presented as fold increases. Data are mean±standard error of the mean (n=6 per group).Dapa = dapagliflozin; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; IL = interleukin; LPS = lipopolysaccharide; NF-κB = nuclear factor-kappa B; RT-PCR = reverse transcription-polymerase chain reaction; TLR4 = Toll-like receptor 4;TNF = tumor necrosis factor.*p<0.05 vs. LPS group.


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Chi Young Shim
Korean Circ J. 2020;50(12):1051-1061.    doi: 10.4070/kcj.2020.0338.


Reference

1. Murray CJ, Lopez AD. Measuring the global burden of disease. N Engl J Med. 2013; 369:448–457. PMID: 23902484.
2. Libby P. Inflammation in atherosclerosis. Nature. 2002; 420:868–874. PMID: 12490960.
3. Glass CK, Witztum JL. Atherosclerosis. The road ahead. Cell. 2001; 104:503–516. PMID: 11239408.
4. Allahverdian S, Pannu PS, Francis GA. Contribution of monocyte-derived macrophages and smooth muscle cells to arterial foam cell formation. Cardiovasc Res. 2012; 95:165–172. PMID: 22345306.
5. Rieg T, Masuda T, Gerasimova M, et al. Increase in SGLT1-mediated transport explains renal glucose reabsorption during genetic and pharmacological SGLT2 inhibition in euglycemia. Am J Physiol Renal Physiol. 2014; 306:F188–93. PMID: 24226519.
6. Ferrannini E, Solini A. SGLT2 inhibition in diabetes mellitus: rationale and clinical prospects. Nat Rev Endocrinol. 2012; 8:495–502. PMID: 22310849.
7. Heerspink HJ, Perkins BA, Fitchett DH, Husain M, Cherney DZ. Sodium glucose cotransporter 2 inhibitors in the treatment of diabetes mellitus: cardiovascular and kidney effects, potential mechanisms, and clinical applications. Circulation. 2016; 134:752–772. PMID: 27470878.
8. Bhartia M, Tahrani AA, Barnett AH. SGLT-2 inhibitors in development for type 2 diabetes treatment. Rev Diabet Stud. 2011; 8:348–354. PMID: 22262072.
9. Abdul-Ghani M, Del Prato S, Chilton R, DeFronzo RA. SGLT2 inhibitors and cardiovascular risk: lessons learned from the EMPA-REG OUTCOME study. Diabetes Care. 2016; 39:717–725. PMID: 27208375.
10. Henry RR, Rosenstock J, Edelman S, et al. Exploring the potential of the SGLT2 inhibitor dapagliflozin in type 1 diabetes: a randomized, double-blind, placebo-controlled pilot study. Diabetes Care. 2015; 38:412–419. PMID: 25271207.
11. Han JH, Oh TJ, Lee G, et al. The beneficial effects of empagliflozin, an SGLT2 inhibitor, on atherosclerosis in ApoE −/− mice fed a western diet. Diabetologia. 2017; 60:364–376. PMID: 27866224.
12. Terasaki M, Hiromura M, Mori Y, et al. Amelioration of hyperglycemia with a sodium-glucose cotransporter 2 inhibitor prevents macrophage-driven atherosclerosis through macrophage foam cell formation suppression in type 1 and type 2 diabetic mice. PLoS One. 2015; 10:e0143396. PMID: 26606676.
13. Leng W, Ouyang X, Lei X, et al. The SGLT-2 inhibitor dapagliflozin has a therapeutic effect on atherosclerosis in diabetic ApoE−/− mice. Mediators Inflamm. 2016; 2016:6305735. PMID: 28104929.
14. Kim JS, Lee SG, Oh J, et al. Development of advanced atherosclerotic plaque by injection of inflammatory proteins in a rabbit iliac artery model. Yonsei Med J. 2016; 57:1095–1105. PMID: 27401639.
15. Lee SG, Lee SJ, Thuy NV, et al. Synergistic protective effects of a statin and an angiotensin receptor blocker for initiation and progression of atherosclerosis. PLoS One. 2019; 14:e0215604. PMID: 31050669.
16. Tsunoda F, Asztalos IB, Horvath KV, Steiner G, Schaefer EJ, Asztalos BF. Fenofibrate, HDL, and cardiovascular disease in type-2 diabetes: the DAIS trial. Atherosclerosis. 2016; 247:35–39. PMID: 26854974.
17. Kohan DE, Fioretto P, Tang W, List JF. Long-term study of patients with type 2 diabetes and moderate renal impairment shows that dapagliflozin reduces weight and blood pressure but does not improve glycemic control. Kidney Int. 2014; 85:962–971. PMID: 24067431.
18. Marx N, McGuire DK. Sodium-glucose cotransporter-2 inhibition for the reduction of cardiovascular events in high-risk patients with diabetes mellitus. Eur Heart J. 2016; 37:3192–3200. PMID: 27153861.
19. Chinetti-Gbaguidi G, Colin S, Staels B. Macrophage subsets in atherosclerosis. Nat Rev Cardiol. 2015; 12:10–17. PMID: 25367649.
20. Peled M, Fisher EA. Dynamic aspects of macrophage polarization during atherosclerosis progression and regression. Front Immunol. 2014; 5:579. PMID: 25429291.
21. Rőszer T. Understanding the mysterious M2 macrophage through activation markers and effector mechanisms. Mediators Inflamm. 2015; 2015:816460. PMID: 26089604.
22. Pourcet B, Pineda-Torra I. Transcriptional regulation of macrophage arginase 1 expression and its role in atherosclerosis. Trends Cardiovasc Med. 2013; 23:143–152. PMID: 23375628.
23. McKellar GE, McCarey DW, Sattar N, McInnes IB. Role for TNF in atherosclerosis? Lessons from autoimmune disease. Nat Rev Cardiol. 2009; 6:410–417. PMID: 19421244.
24. Moriya J. Critical roles of inflammation in atherosclerosis. J Cardiol. 2019; 73:22–27. PMID: 29907363.
25. Xu C, Wang W, Zhong J, et al. Canagliflozin exerts anti-inflammatory effects by inhibiting intracellular glucose metabolism and promoting autophagy in immune cells. Biochem Pharmacol. 2018; 152:45–59. PMID: 29551587.
26. Mancini SJ, Boyd D, Katwan OJ, et al. Canagliflozin inhibits interleukin-1β-stimulated cytokine and chemokine secretion in vascular endothelial cells by AMP-activated protein kinase-dependent and -independent mechanisms. Sci Rep. 2018; 8:5276. PMID: 29588466.
27. Yan ZQ. Regulation of TLR4 expression is a tale about tail. Arterioscler Thromb Vasc Biol. 2006; 26:2582–2584. PMID: 17110607.
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