Glibenclamide ameliorates transplant-induced arteriosclerosis and inhibits macrophage migration and MCP-1 expression

Journal Pre-proof Glibenclamide ameliorates transplant-induced arteriosclerosis and inhibits macrophage migration and MCP-1 expression

Yanqiang Zou, Cheng Zhou, Heng Xu, Jizhang Yu, Ping Ye, Hao Zhang, Shanshan Chen, Jing Zhao, Sheng Le, Jikai Cui, Lang Jiang, Jie Wu, Jiahong Xia PII:

S0024-3205(19)31069-0

DOI:

https://doi.org/10.1016/j.lfs.2019.117141

Reference:

LFS 117141

To appear in:

Life Sciences

Received date:

12 September 2019

Revised date:

20 November 2019

Accepted date:

1 December 2019

Please cite this article as: Y. Zou, C. Zhou, H. Xu, et al., Glibenclamide ameliorates transplant-induced arteriosclerosis and inhibits macrophage migration and MCP-1 expression, Life Sciences(2019), https://doi.org/10.1016/j.lfs.2019.117141

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© 2019 Published by Elsevier.

Journal Pre-proof

Glibenclamide ameliorates transplant-induced arteriosclerosis and inhibits macrophage migration and MCP-1 expression Yanqiang Zoua,1, M.D., Cheng Zhoua,1, M.D., Heng Xua,1, M.D., Jizhang Yua, M.D., Ping Yeb, M.D., Hao Zhanga, M.D., Shanshan Chena, M.D., Jing Zhaoa, M.D., Sheng Lea, M.D., Jikai Cuia, M.M., Lang Jianga, M.M., Jie Wua,*, M.D., Jiahong Xiaa,*, M.D.

a

Department of Cardiothoracic Surgery, The Union Hospital, Tongji Medical College,

b

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Huazhong University of Science and Technology, Wuhan 430022, China Department of Cardiology, The Central Hospital of Wuhan, Tongji Medical College,

These authors contributed equally to this work.

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1

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Huazhong University of Science and Technology, Wuhan 430022, China

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*Correspondence to: Jiahong Xia or Jie Wu, in the Department of Cardiothoracic Surgery, The Union Hospital, Tongji Medical College, Huazhong University of Science and 1277

Jiefang

Avenue,

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Technology,

Wuhan

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[email protected] or [email protected]

1

430022,

China.

E-mail:

Journal Pre-proof Author contributions JX and JW conceived and designed the experiments; YZ, HZ, and SC drafted the manuscript; YZ, CZ, and HX performed the experiments; JC, LJ, and SL established the animal models; PY, JY, and JZ analyzed the data. All authors have read the final version of this manuscript and approved this submission.

Conflict of interest statement

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The authors declare that there is no conflict of interest in this work.

Financial support statement

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This study was supported in part by grants from the National Natural Science Foundation of

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China (#81730015, #81571560, #81701585, and #81570325) and the Natural Science Fund

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of Hubei Province (#2017CFB357 and #2019AAA032).

Consent for publication

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ur

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Consent to publish has been obtained from the participants.

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Journal Pre-proof Abstract Aims: Glibenclamide, a diabetes mellitus type 2 medication, has anti-inflammatory and autoimmune properties. This study investigated the effects of glibenclamide on transplant-induced arteriosclerosis as well as the underlying molecular events. Methods: Male C57Bl/6 (H-2b) and BALB/c (H-2d) mice were used for aorta transplantation. We used hematoxylin and eosin (HE) and Elastic Van Gieson (EVG) staining for histological assessment, and qRT-PCR and ELISA to measure mRNA and protein levels. Mouse

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peritoneal macrophages were isolated for lipopolysaccharide (LPS) stimulation and glibenclamide treatment followed by ELISA, Western blot, and Transwell assays. Results: Glibenclamide inhibited transplant-induced arteriosclerosis in vivo. Morphologically,

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glibenclamide reduced inflammatory cell accumulation and collagen deposition in the aortas. At the gene level, glibenclamide suppressed aortic cytokine mRNA levels, including

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interleukin-1β (IL-1β; 10.64 ± 3.19 vs. 23.77 ± 5.72; P < 0.05), tumor necrosis factor-α

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(TNF-α; 4.59 ± 0.78 vs. 13.89 ± 5.42; P < 0.05), and monocyte chemoattractant protein-1 (MCP-1; 202.66 ± 23.44 vs. 1172.73 ± 208.80; P < 0.01), while IL-1β, TNF-α, and MCP-1

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levels were also reduced in the mouse sera two weeks after glibenclamide treatment (IL-1β, 39.40 ± 13.56 ng/ml vs. 78.96 ± 9.39 ng/ml; P < 0.01; TNF-α, 52.60 ± 13.00 ng/ml vs.

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159.73 ± 6.76 ng/ml; P < 0.01; and MCP-1, 56.60 ± 9.07 ng/ml vs. 223.07 ± 36.28 ng/ml; P <

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0.001). Furthermore, glibenclamide inhibited macrophage expression and secretion of inflammatory factors in vitro through suppressing activation of the nuclear factor-κB (NF-κB) pathway and MCP-1 production. Conclusion: Glibenclamide protected against aorta transplantation-induced arteriosclerosis by reducing inflammatory factors in vivo and inhibited macrophage migration and MCP-1 production in vitro. Keywords: Mouse model of graft arteriosclerosis, glibenclamide, NF-κB, MCP-1

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Journal Pre-proof 1. Introduction Organ transplant-induced arteriosclerosis, characterized by concentric stenosis-induced occlusion of blood vessels, is a major challenge for long-term survival of patients with organ transplantation (1). Mechanistically, organ transplantation-induced arteriosclerosis could be due to acute innate and acquired immune responses, which in turn damage endothelial cells and promote accumulation of smooth muscle cells, leading to arteriosclerosis development (2, 3). For example, a variety of immune responses and related factors are involved in lesion

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establishment, such as macrophages, dendritic cells, alloantibodies, pro-inflammatory factors, cell adhesion molecules, as well as other non-immune factors, such as recipient insulin resistance, hyperlipidemia, or infection of

cytomegalovirus (1, 4-6). Transplant

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arteriosclerosis (TA) is a common problem in organ transplants, especially in heart

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transplants (7). Heart transplantation is currently the only effective treatment for cardiac dysfunction and failure (8). Although the 5-year survival rate of heart-transplanted patients

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has been significantly extended to 72.5% with the administration of immunosuppressants (9), TA is still a significant barrier to long-term survival of patients (10, 11). Previous studies

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revealed that macrophages could promote TA development by regulating various chemokines and growth factors (12-14). However, novel strategies to effectively control TA development

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and progression are urgently needed.

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Glibenclamide has been clinically used to treat type 2 diabetes mellitus as a sulfonylureas drug, but has been recently shown to possess anti-inflammatory and neuroprotective properties (15). Glibenclamide typically has very limited side effects (nausea and heartburn), although it has been reported to induce serious side effects, such as angioedema and low blood sugar [https://www.drugs.com/monograph/glyburide.html]. Recently, glibenclamide was demonstrated to inhibit neutrophil migration and chemotaxis to block ATP-sensitive potassium channels (16) and to attenuate lipopolysaccharides (LPS)-induced myocardial injury through inhibiting the NLRP3 inflammasome (17). In vivo, glibenclamide mitigated atherosclerosis by suppressing ATP-sensitive potassium channels in atherosclerotic lesions in a mouse model (18). A previous clinical retrospective study also demonstrated that diabetes 4

Journal Pre-proof patients with melioidosis had a better prognosis after taking glibenclamide, which could be related to its anti-inflammatory activities (19). Glibenclamide could also relieve autoimmune diseases, including multiple sclerosis, by inhibiting transient receptor potential cation channel subfamily M member 4 activity (20). In this study we investigated the effects of glibenclamide on transplant-induced arteriosclerosis and explored the underlying molecular mechanism using a mouse TA model with allogeneic arterial grafts. We assessed intima-media (I/M) ratios and intimal hyperplasia

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in mouse aortas, expression of interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and monocyte chemoattractant protein-1 (MCP-1), aortic inflammatory cell accumulation, and collagen deposition. We isolated macrophages from mice for LPS

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stimulation to determine the effect of glibenclamide on inflammation-related gene expression

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and macrophage migration. Our study provides novel information regarding the use of

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glibenclamide to protect against TA in organ transplantation patients.

2.1 Animals

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2. Material and methods

Male C57Bl/6 (H-2b) and BALB/c (H-2d) mice aged 8 to 12 weeks were obtained from

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Charles River (Beijing, China). The animal protocol of this study was approved by the

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Institutional Animal Care and Use Committee (IACUC) of Huazhong University of Science and Technology (Wuhan, China) and followed the Guidelines of the Care and Use of Laboratory Animals issued by the Chinese Council on Animal Research.

2.2 Abdominal aortic transplantation and animal treatment Abdominal aorta transplantation was performed according to a previous study (21). Donor aortas from C57Bl/6 (H-2b) and BALB/c (H-2d) mice were transplanted into C57Bl/6 (H-2b) mice. In brief, donor abdominal aortas were isolated and the branch vessels were ligated, while the recipient vessels were cut from the midsection using microscopic scissors. The recipient and donor vessels were anastomosed with a cuff suture. On the second day after 5

Journal Pre-proof aorta transplantation, the recipient mice were intraperitoneally injected with 10 μg of glibenclamide or vehicle (sterile PBS of equal volume) daily. The mice were sacrificed 4 or 8 weeks following transplantation for histological and molecular analyses of the aortic grafts.

2.3. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) Total cellular RNA was isolated from whole aortic grafts or peritoneal macrophages using the Trizol reagent (Cat. #9108Q, Takara, Beijing, China) and reverse transcribed into cDNA

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using the PrimeScript™ RT Master Mix (Cat. #RR036A; Takara) according to the manufacturer’s instructions. qPCR was performed using the Real SYBR kit (Cat. #CW0760M, CWBIO, Beijing, China) in an ABI Prism 7500 instrument (Applied

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Biosystems, Foster City, CA, USA). The relative levels of Il-1b, Il-6, Mcp1, Tnf-α, intracellular adhesion molecule-1 (Icam1), and Rantes mRNA were normalized to Gapdh

three

times.

The

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repeated

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mRNA and quantified using the 2-∆∆Ct method. Each sample was measured in triplicate and primer

sequences

were

Il-1b,

5'-GCAACTGTTCCTGAACTCAACT-3' and 5'-ATCTTTTGGGGTCCGTCAACT-3'; Il-6,

Mcp1,

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5'-TAGTCCTTCCTACCCCAATTTCC-3'

and

5'-TTGGTCCTTAGCCACTCCTTC-3';

5'-TTAAAAACCTGGATCGGAACCAA-3'

Tnf-α

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5'-GCATTAGCTTCAGATTTACGGGT-3';

and

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5'-CCCTCACACTCAGATCATCTTCT-3' and 5'-GCTACGACGTGGGCTACAG-3'; Icam1, 5'-GTGATGCTCAGGTATCCATCCA-3' and 5'-CACAGTTCTCAAAGCACAGCG-3'; and Rantes, 5'-CATATGGCTCGGACACCA-3' and 5'-ACACACTTGGCGGTTCCT-3'.

2.4 Histology and immunohistochemistry Abdominal aortic grafts were harvested from mice 4 and 8 weeks after transplantation, fixed in 4% paraformaldehyde solution over night, and then processed for paraffin-embedding and sectioning into 5-µm serial sections for morphometric and immunohistochemical analyses. Hematoxylin and eosin (HE) and Elastic Van Gieson (EVG) staining were performed according to our routine laboratory protocols, and the I/M ratios and intimal hyperplasia area 6

Journal Pre-proof were measured using the ImageJ software (National Institute of Heath, Bethesda, MD, USA). We also performed immunohistochemistry to assess cell proliferation and inflammatory cell infiltration in the aortic allografts using the following antibodies according to the manufacture’s protocols: anti-alpha smooth muscle actin (Cat. #GB13044; Servicebio, Wuhan, China), anti-CD3 (Cat. #GB13014; Servicebio), rabbit polyclonal antibody (pAb) anti-F4/80 (Cat. #GB11027; Servicebio), anti-collagen I (Cat. #GB13022-3; Servicebio), and anti-MCP-1 (Cat. #GB11199; Servicebio). The mean optical densities and positive cells were

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reviewed and scored under a light microscope at a magnification of 400x and quantified using the ImageJ software.

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2.5 Isolation of mouse peritoneal macrophages and in vitro treatment

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Mouse primary peritoneal macrophages were isolated according to a previous study (22). Briefly, male C57Bl/6 mice aged 8 - 12 weeks were injected with 1 ml of 5% thioglycollate

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broth using a 1 ml insulin syringe. Three days later, mouse primary peritoneal macrophages were harvested with three repeating lavages. Peritoneal macrophages were then incubated at

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37°C in a humidified incubator at 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum (FBS) (Gibco, Gaithersburg, MD, USA),

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100 units/ml penicillin, and 100 µg/ml streptomycin (Cat. #15140163; Gibco). For our

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experiments, peritoneal macrophages were pretreated with glibenclamide or vehicle (PBS) 30 min before addition of 500 ng/ml LPS (Cat. #S1732; Beyotime Biotechnology, Shanghai, China).

2.6 Enzyme-linked immunosorbent assay (ELISA) The concentrations of TNF-α, MCP-1, and IL-1β in peritoneal macrophage-cultured supernatants and mouse serum samples two weeks after transplantation were assessed using ELISA kits from Dakewe Biotech Co., Ltd. (Shenzhen, China) according to the manufacturer’s protocols. Absorbance was recorded at 450 nm using a microplate reader (Thermo Scientific). Each experiment was done in triplicate and repeated three times. 7

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2.7 Western blot analysis Total protein from peritoneal macrophages was extracted using the radioimmunoprecipitation assay buffer (RIPA buffer; Cat. #P0013B, Beyotime Biotechnology, Shanghai, China), and the protein concentration was quantified using the bicinchoninic acid (BCA) protein assay kit (CWBIO) according to the manufacturers’ protocols. An equal amount of protein was separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

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gels and electrically transferred onto a polyvinylidene fluoride membrane. For Western blot analysis, the membranes were first blocked in 5% bovine serum albumin (BSA) in Tris-based saline-Tween 20 (TBS-T) for 1 - 2 h at room temperature. Next, the membranes were

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incubated with different primary antibodies, including rabbit monoclonal anti-MEK1/2 (Cat. #AF1057; Beyotime Biotechnology; 1:1,000), rabbit monoclonal anti-p38 (Cat. #AF1111;

Biotechnology;

1:1,000),

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Beyotime

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Beyotime Biotechnology; 1:1,000), rabbit polyclonal anti-NF-κB p65 (Cat. #AF0246;

anti-phospho-MAP2K1-S217/MAP2K2-S221

(Cat.

rabbit #AP0209;

ABclonal;

polyclonal 1:1,000),

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anti-MCP1 (Cat. #66272-Ig, Proteintech, Wuhan, China; 1:1,000), anti-phospho-p38 MAPK (Cat. #AM063; Beyotime Biotechnology; 1:1,000), anti-phospho-NF-κB p65 (Cat. #AN371;

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Beyotime Biotechnology; 1:1,000), and anti-HRP-GAPDH (Cat. #HRP-60004; Beyotime

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Biotechnology; 1:10,000), followed by a goat anti-rabbit or goat anti-mouse secondary antibody (Beyotime Biotechnology). Protein bands were detected using BeyoECL Plus (Cat. #P0018S; Beyotime Biotechnology) according to the manufacturer’s instructions. Each experiment was repeated three times.

2.8 Cell Counting Kit-8 Primary vascular smooth muscle cells (1×105 cells/ml) were seeded in a serum-free medium for 24 hours, then treated with 20% serum from the mouse of transplantation atherosclerosis for two weeks. Cell counting kit-8 (CCK-8; Cat. #C0037 Beyotime Biotechnology) mixed

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Journal Pre-proof with medium was used for cell viability assay. And the OD value was recorded at 450nm on designated time. Each experiment was repeated three times.

2.9 Flow cytometry Animals were anesthetized at 2 weeks after transplantation and then 150μl peripheral blood and 150mg spleen tissues were harvested for lysing red blood cells in samples. Single-cell suspensions were was centrifuged at 1500rpm for 4 min at 4 °C and then cell preparations

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were stained using the following fluorochrome-conjugated antibodies:Purified anti-mouse CD16/32 (Biolegend 101301) CD3e-PE (Biolegend 100308), CD19-APC/Cy7 (Biolegend 115529), Ly6C-FITC (eBioscience 2106875), Ly6G -PE Cy7 (Biolegend 127617),

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CD45-PerCP Cy5.5 (Biolegend 103132), NK1.1-Pacific Blue (Biolegend 108721),

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CD11b-APC-Cy7 (Biolegend 101225), CD11c-APC (Biolegend 117309), B220-FITC (Biolegend 103206) and F4/80-APC (Biolegend 123115). Dead cells were excluded from

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some analysis by using Zombie Dyes (Biolegend 77143). Data were collected on

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LSRFortessa X-20 (BD Biosciences) and analyzed using Flowjo software (Tree Star).

2.10 Isolation of primary vascular smooth muscle cells and in vitro treatment

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Vascular smooth muscle cells were harvested from the descending thoracic aorta. In brief,

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aortas were first cut into small pieces and then digested with type II collagenase (Worthington Biochemical Corporation) at 37°C for 1 h, as previously described (23). The cells were cultured in DMEM containing 20% FBS. Cells were used at passages 4 - 6 for subsequent experiments.

2.11 Transwell assay Cell migration capacity was assessed using a Transwell chamber with an 8-μm pore filter (Corning; Corning, NY, USA). In brief, macrophages (5 x 105/well) were seeded into the upper chamber in DMEM only, while the lower chamber was filled with conditioned medium from the LPS-stimulated macrophages after 24-h glibenclamide or vehicle treatment. Primary 9

Journal Pre-proof vascular smooth muscle cells (5 x 105/well) were also placed into the upper chamber in DMEM only, while the lower chamber was filled with DMEM containing 20% mouse serum from mice after transplantation-induced atherosclerosis and 2-weeks of treatment with or without glibenclamide. Cells on the top filter were removed after 24 h, and the cells on the bottom filters were fixed in 4% paraformaldehyde solution for 10 min at room temperature and then stained with 0.05% crystal violet solution. The number of cells that migrated across

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the filter was counted in five random fields at a magnification of ×200 under a microscope.

2.12 Statistical analysis.

All data were collected from three or more independent experiments and are summarized as

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mean ± standard error (SEM). The unpaired Student’s t-test and one-way ANOVA followed

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by Bonferroni correction were performed to determine statistical significance using the SPSS 19.0 software (Chicago, IL, USA) or GraphPad Prism 7.0a (GraphPad Software, Inc., San

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3. Results

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Diego, CA) for Mac OS X. A P < 0.05 was considered statistically significant.

3.1 Glibenclamide suppresses transplant-induced arteriosclerosis in vivo

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We evaluated the effect of glibenclamide on the pathogenesis of chronic arterial allograft

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rejection using our mouse vascular graft arteriosclerosis model. HE and EVG staining (Fig. 1A) showed that glibenclamide treatment attenuated the degree of vascular stenosis (Fig. 1B) and intimal hyperplasia (Fig. 1C) compared to the vehicle treatment group. Moreover, compared to the vehicle group, glibenclamide treatment reduced the I/M ratio (1.46 ± 0.42 vs. 2.39 ± 0.24 four weeks after treatment, P < 0.01; 2.47 ± 0.52 vs. 3.31 ± 0.61 eight weeks after treatment; P < 0.05) and decreased neointimal area (23,780 ± 4,595 μm2 vs. 46,712 ± 3,417 μm2; P < 0.001 four weeks of treatment; 37,468 ± 3,538 μm2 vs. 80,548 ± 2,398 μm2; P < 0.05). However, blood glucose levels were not significantly changed post-operatively after treatment between the two groups (Fig. 1D and Fig. S1).

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Journal Pre-proof 3.2 Glibenclamide reduces aortic inflammatory cell accumulation and collagen deposition To investigate the effect of glibenclamide on chronic inflammatory cell infiltration, we assessed the number of CD3-positive T cells and F4/80-positive macrophages in the mouse aortas using immunohistochemistry (Fig. 2A). We found that the number of CD3 positive cells (Fig. 2B) was not statistically different in the glibenclamide treatment group four weeks after graft transplantation compared to the control group (8.9 ± 2.2% vs. 9.0 ± 1.7%; P = 0.38). However, at four weeks, glibenclamide reduced macrophage infiltration compared to

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the control group (Fig. 2C; 15.1 ± 2.2% vs. 9.2 ± 1.4%; P < 0.01). To further assess the effect of glibenclamide on smooth muscle proliferation, we measured α-SMA density (Fig. 2D; 11 ± 2% vs. 29 ± 8%; P < 0.01), and collagen fiber deposition (Fig. 2E; 0.104 ± 0.020 vs. 0.346

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± 0.041; P < 0.001), which was consistent with the inhibition of glibenclamide on smooth

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muscle cell migration in vitro (Fig. S2A). In addition, the levels of macrophages in the peripheral blood and spleen were lower in the glibenclamide-treated group compared to the

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vehicle group, while the other cell subpopulations were not significantly difference (Data

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shown in Fig. S3).

arteriosclerosis model

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3.3 Glibenclamide inhibits secretion of aortic cytokines in mouse vascular graft

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We next assessed levels of pathogenic inflammatory factors (Il-1b, Il-6, Tnf-α, Icam1, Mcp1, and Rantes) in the graft vessels after two week-glibenclamide treatment (Fig. 3). After calibrating the data with the results of the isograft, we found a significant downregulation of Il-1b (10.64 ± 3.19 vs. 23.77 ± 5.72; P < 0.05), Tnf-α (4.59 ± 0.78 vs. 13.89 ± 5.42; P < 0.05), and Mcp1 (202.66 ± 23.44 vs. 1172.73 ± 208.80; P < 0.01). Simultaneously, the secretion of MCP-1, IL-1β, and TNF-α in the mouse sera two weeks after glibenclamide treatment was also suppressed (IL-1β, 39.40 ± 13.56 ng/ml vs. 78.96 ± 9.39 ng/ml; P < 0.01; TNF-α, 52.60 ± 13.00 ng/ml vs. 159.73 ± 6.76 ng/ml; P < 0.01; and MCP-1, 56.60 ± 9.07 ng/ml vs. 223.07 ± 36.28 ng/ml; P < 0.001).

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Journal Pre-proof 3.4 Glibenclamide inhibits expression and secretion of inflammatory factors in peritoneal macrophages, as well as macrophage migration in vitro We next assessed the effect of glibenclamide on activated peritoneal macrophages in vitro. We isolated macrophages from mice and stimulated them with LPS (500 ng/ml) and glibenclamide treatment (Fig. 4). Six hours of LPS stimulation induced expression of Mcp1, Il-1b, Tnf, and Icam1, whereas glibenclamide treatment inhibited expression of Mcp1 (15.63 ± 2.09 vs. 65.30 ± 4.06; P < 0.001), Il-1b (4.84 ± 1.05 vs. 34.15 ± 4.1; P < 0.001), Tnf-α (5.55

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± 1.15 vs. 3.01 ± 0.82; P < 0.05), and Icam1 (3.01 ± 0.50 vs. 4.9 ± 0.42; P < 0.01). Our ELISA data confirmed the changes in expression of these factors in the cell culture supernatants (Fig. 4E - G). In addition, our Transwell assay data confirmed that

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glibenclamide treatment suppressed migration of LPS-activated macrophages (131 ± 8 vs.

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224 ±9, P < 0.01; Fig. 4H - I).

activated macrophages in vitro

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3.5 Glibenclamide suppresses activation of the NF-κB pathway and MCP-1 production in

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The NF-κB and MAPK signaling pathways have been related to inflammatory factor secretion and macrophage migration (22). We thus assessed the effects of glibenclamide on

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activation of these pathways in vitro (Fig. 5A). Our results showed that glibenclamide

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suppressed phosphorylation of p65 (Fig. 5B), but did not change the level of phosphorylated MEK and p38 (Fig. 5B-C) in the isolated macrophages. Moreover, expression of MCP-1 was dose-dependently inhibited by 24-h treatment of macrophages with glibenclamide (Fig. 5E F). Indeed, expression of MCP-1 was lower in the glibenclamide-treated group compared to the control group (Fig. 5G - H; 0.33 ± 0.23 vs. 0.43 ± 0.21; P < 0.01).

4. Discussion In the current study, we assessed the effects of glibenclamide on transplant-induced arteriosclerosis and investigated the underlying molecular mechanism using both a mouse model and cultured macrophages. We found that glibenclamide ameliorated development of 12

Journal Pre-proof transplant-induced arteriosclerosis in vivo and inhibited LPS-activated macrophage migration and MCP-1 expression in vitro. Morphologically, glibenclamide reduced aortic macrophage accumulation and collagen deposition, and reduced cytokine mRNA levels in the aortas and protein in the blood. Furthermore, glibenclamide inhibited expression and secretion of inflammatory factors, inhibited NF-κB pathway activation, and reduced MCP-1 expression in macrophages in vitro. Thus, our current study demonstrated that glibenclamide protects against transplant-induced arteriosclerosis by reducing inflammation in vivo and in vitro.

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Organ transplantation-induced arteriosclerosis is a major cause of allograft failure (1). A recent review summarized novel targets to prevent chronic rejection after thoracic organ transplantation (23). Both immune and non-immune responses are involved in graft

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arteriosclerosis after organ transplantation (3, 4). In the transplantation-induced

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atherosclerosis model, intimal thickening is caused by vascular damage that triggers migration of inflammatory cells into injured sites of the vessels. Cytokines and growth

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factors (such as PDGF-BB, TNF-α, IL-6, MCP-1) produced by activated inflammatory cells and damaged vascular cells stimulate smooth muscle cell migration into the intima where the

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cells proliferate and facilitate aggregation of additional inflammatory cells (24). Previous studies have reported that macrophages play an important role in TA progression. For

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example, Abele et al., showed that clopidogrel was able to alleviate TA by reducing

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infiltrating dendritic cells and macrophages (25), while clearance of invaded macrophages in transplanted organs by the chemokines receptor antagonist met-RANTES (26) and deletion of ccr1 (27) or ccr5 (28) prolonged graft survival. In the current study, we investigated the TLR4 activation pathway to assess the effects of glibenclamide on inflammatory reactive macrophages in vitro. We measured levels of IL-1β, TNF-α, and MCP-1 in the aortas and mouse sera from mice following allograft and in LPS-activated macrophages. We found that glibenclamide treatment decreased the levels of these cytokines. Indeed, previous studies showed that IL-6, IL-17, IL-1β, and TNF-α significantly activated porcine aortic endothelial cells and promoted inflammation and coagulation in response to xenografts (29), while early post-transplant inflammation enhanced alloimmunity and chronic human lung allograft 13

Journal Pre-proof rejection (30). There is also evidence showing that IL-8, IL-10, MCP-1, and MCP-3 were upregulated in peripheral blood monocytes in stable lung transplant recipients (31). Taken together, a variety of immune factors underlie organ transplantation-induced arteriosclerosis. Glibenclamide is a common hypoglycemic drug used clinically as either a monotherapy or a combination therapy with other agents, diet, and exercise. Glibenclamide dosage is typically administered as > 10 mg daily (initially 2.5 -5 mg daily) as a conventional formulation, or > 6 mg as a micronized glyburide [Pharmacia & Upjohn Company. Micronase (glyburide)

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prescribing information. Kalamazoo, MI; 2002]. Recently, glibenclamide has been shown to regulate inflammation (32). For example, Liao et al., reported that glibenclamide was able to inhibit activation of the NLRP3 inflammasome in hypoxia-injured mice (33), while Makar et

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al., showed that glibenclamide suppressed the Sur1-Trpm4 channels and significantly

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ameliorated disease progression in experimental autoimmune encephalomyelitis by improving neurological functions (34). In atherosclerosis, glibenclamide has been

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demonstrated to inhibit inflammation and stabilize plaques by reducing ATP-sensitive potassium channels in macrophages (18). In our current study, we found that glibenclamide

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suppressed intimal hyperplasia and collagen deposition in grafted blood vessels without significantly affecting blood glucose levels. However, further study is needed to precisely

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assess the optimal dosage of glibenclamide in such a setting. In human diabetes,

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glibenclamide can be prescribed at > 10 mg daily in a conventional formulation, but in our current study we intraperitoneally injected 10 μg of glibenclamide or PBS as a negative control daily into mice. At this dosage, mice did not show significant changes in blood glucose levels, indicating that 10 μg of glibenclamide is a clinically achievable dosage for organ transplant patients. In TA pathology, recruitment of lymphocytes and macrophages could promote damage to allografted vessels, further leading to a cytokine-rich environment that promotes migration and accumulation of vascular smooth muscle cells (35-37). A previous study revealed that removal or reduction of macrophage recruitment into the lesion sites could slow TA progression (25). In contrast, proliferation and migration of vascular smooth muscle cells are 14

Journal Pre-proof central events in TA development and progression (38), while collagen synthesis was associated with smooth muscle cell migration. In vitro inhibition of collagen synthesis has been shown to suppress smooth muscle cell spread and migration (39). In our current study, although glibenclamide had no effect on smooth muscle cell proliferation, it was able to inhibit migration in vitro and inhibit neointimal deposition of collagen in vivo. We also showed that glibenclamide antagonized LPS-induced macrophage activation, cytokine expression, and macrophage migration, which was associated with glibenclamide

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inhibition of MCP-1 expression and secretion. Similar findings have been reported for TNF-α and IL-1β (40-42). The NF-κB and MAPK pathways are the major downstream signaling pathways in TLR4 receptor mediated immune responses in activated macrophages (43-45).

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LPS is a lipoglycan and endotoxin derived from the outer membrane of Gram-negative bacteria.

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LPS can induce immune responses in animals, including humans, by binding to the CD14/TLR4/MD2 receptor complex in monocytes, dendritic cells, macrophages, and B

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lymphocytes (Raetz CR, Whitfield C. Lipopolysaccharide endotoxins. Annu. Rev. Biochem. 71: 635–700, 2002). In the current study, we utilized LPS to activate macrophages to assess the

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effects of glibenclamide in vitro. We showed that LPS was able to induce macrophage activation, cytokine expression, and macrophage migration, whereas glibenclamide inhibited

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p-p65 activation, the key molecule in the NF-κB pathway. However glibenclamide had no

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effect on p-p38 and p-MEK activation in macrophages, which is in contrast to a study by Ling et al. (18). This inconsistency may be due to our use of primary peritoneal macrophages. In addition, MCP-1 is the key chemokine that regulates macrophage migration and infiltration in TA (46-48). Saiura et al., revealed that anti-MCP-1 gene therapy controlled TA (48). Our previous study also showed that loss of miRNA-155 in the bone marrow downregulated MCP-1 expression and inhibited smooth muscle cell migration, mitigating TA (49). Here, we found that glibenclamide inhibited MCP-1 expression in LPS-activated primary macrophages and in allograft lesions in mice, which in turn decreased macrophage infiltration in the TA lesions.

15

Journal Pre-proof Taken together, our current study demonstrated that glibenclamide, a frequently clinically used hypoglycemic agent, reduced allograft production and secretion of TNF-α, IL-1β, and MCP-1 in mice and that recruitment of macrophages in the TA lesions was partly explained by glibenclamide inhibition of TA development and progression. However, our current study only explored the potential effect of glibenclamide on macrophage function, and future studies are needed to validate the effects of glibenclamide on TA alleviation in the clinic.

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Journal Pre-proof Figure legends

Figure

1.

Glibenclamide

ameliorated

allograft

arteriosclerosis

development.

(A)

Hematoxylin and eosin (HE) and Elastin van Giesson (EVG) staining. Images show representative allogeneic aortic specimens after four or eight weeks of vehicle or glibenclamide treatment in mice after transplantation. All scale bars, 200 µm. (B) Graphs show quantitative analysis of intima to media ratio (I/M). (C) Graphs show quantitative

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analysis of neointimal area (µm2). Original magnification, 100x. Data are presented as mean ± SEM. (D) Record changes in blood glucose at 4 and 8 weeks. (n = 6 - 7 mice per group *P < 0.05, **P < 0.01, and ***P < 0.001 analyzed using the unpaired two-tailed Student’s

re

-p

t-test. NS, not significant.)

Figure 2. Glibenclamide reduced inflammatory cell accumulation and collagen deposition.

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(A) Immunohistochemistry. Representative sections of allogeneic aortas stained for T lymphocytes (CD3+ cells/hpfs), macrophages (F4/80+ cells/hpfs), α-SMA, and collagen I on

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day 28 after transplantation (L, the lumen with a magnification of 400s). (B - E) Graphs show quantitative analysis of the percentages of CD3+ cells/hpfs, F4/80+ cells/hpfs, α-SMA, and

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Collage I per area. Data show a significant reduction in F4/80-positive cells (P < 0.01) in the

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allogeneic aortas in glibenclamide-treated mice. All scale bars, 50 µm. Data are presented as mean values ± SEM (n = 6 - 7 mice per group and 5 cross-sections for per allograft. *P < 0.05, **P < 0.01, and ***P < 0.001 analyzed using the unpaired two tailed Student’s t-test. NS, not significant.

Figure 3. Glibenclamide suppressed cytokine mRNA and protein secretion in mice two weeks after treatment. (A - F) qRT-PCR. mRNA levels in the intragraft were analyzed using qRT-PCR and each diagram shows relative copy numbers of gene expression in untreated control isografts and two-week vehicle- and glibenclamide-treated allografts. (G - I) ELISA. Levels of MCP-1 (G), IL-1β (H), and TNF-α (I) in mouse sera were assessed using ELISA. 21

Journal Pre-proof Data are presented as mean values ± SEM (n = 4 - 5 mice for each group). *P < 0.05, **P < 0.01, and ***P < 0.001 analyzed using the unpaired two tailed Student’s t-test. NS, not significant.

Figure 4. Glibenclamide inhibited expression and secretion of inflammatory factors, as well as migration of LPS-activated peritoneal macrophages. (A - D) qRT-PCR. Relative mRNA levels of Mcp1, Il-1b, Tnf-α, and Icam1 in peritoneal macrophages after pre-treatment with

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glibenclamide (50 μM) or vehicle for 30 min and then stimulated with LPS (500 ng/ml). mRNA levels were assessed by using qRT-PCR. Expression of MCP-1 (E), IL-1β (F), and TNF-α (J) in the supernatants of LPS stimulated peritoneal macrophages pre-treated with

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glibenclamide (50 μM) or vehicle were assessed using ELISA. (H) Wound healing assay. Activated peritoneal macrophages were grown and wounded by scraping with a 200 μl

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pipette tip and then incubated with either glibenclamide (50 μM or 100 μM) or vehicle for 24

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h. Wound closure was measured at 0 h and 24 h. (I) Graphs show quantitative analysis of macrophage migration rate. Data are presented as mean ± SEM. Each experiment was

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repeated three times. *P < 0.05, **P < 0.01, and ***P < 0.001 analyzed using the unpaired

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significant.

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two tailed Student’s t-test and one-way ANOVA with Bonferroni correction. NS, not

Figure 5. Glibenclamide suppressed NF-κB signaling and MCP-1 expression in LPS-activated macrophages. (A) Western blot analysis of p-p65, p-MEK, and p-p38 expression at different time points. (B - D) Graphs show the quantitative analysis of p-p65 (B), p-MEK (C), and p-p38 (D) based on data from three repeated experiments. (E) ELISA. MCP-1 levels were assessed in LPS-activated peritoneal macrophage after glibenclamide treatment. (F) Quantitative data of E. (G) Immunohistochemistry. MCP-1 expression was assessed with an MCP-1 antibody in allografts obtained four weeks after transplantation (Original magnification, 400x; All scale bars, 50 µm). (H) Semi-quantitative data of MCP-1 production (n = 5-6 mice per group and 5 cross-sections for per allograft). *P < 0.05, 22

Journal Pre-proof **P < 0.01, and ***P < 0.001 analyzed using the unpaired two tailed Student’s t-test and

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ur

na

lP

re

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one-way ANOVA with Bonferroni correction.

23

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8