C: Representative Western blots and quantitative data show the levels of eNOS and gp91phox proteins in the thrombotic carotid arteries of both groups

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Increased Dipeptidyl Peptidase-4 Accelerates Chronic Stress-Related Thrombosis in Mouse Carotid Artery Model

*Corresponding author: Prof. Xian Wu Cheng, Department of Cardiology and Hypertension/Heart Center, Yanbian University Hospital, 1327 Juzijie, Yanji 133000, China. Email: [email protected] or [email protected]

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Supplementary Figure Legends

Suppl. Fig. S1. The 2-week restraint stress accelerated the endothelial injury and iron

thrombus formation induced by chloride3 (FeCl3) in the carotid arteries of the mice.

A,B: Representative images and quantitative data show the thrombus areas and the

umbers of CD31⁺ endothelial cells of the thrombotic lesions at 2 weeks of stress combined with thrombus induction. C: Representative Western blots and quantitative data show the levels of eNOS and gp91^phox proteins in the thrombotic carotid arteries of both groups. Results are mean ± SEM. *p<0.01 vs. non-stressed group by Student's t-test or one-way ANOVA followed by Tukey post hoc tests.

Suppl. Fig. S2. Stress increased the circulating platelet numbers as well as the numbers

of leukocytes (Leu) and neutrophils (Neu). A,B: Routine blood tests show the numbers of blood platelets, leukocytes, and neutrophils at 2 weeks of stress combined with thrombus induction. Results are mean ± SEM. *p<0.01 vs. non-stressed group by Student's t-test or one-way ANOVA followed by Tukey post hoc tests.

Suppl. Fig. S3. The 2-week stress increased the expressions of the inflammatory- and

oxidative stress-related genes (i.e., TNF-α, IL-1β, PAI-1, VCAM-1, ICAM-1, MCP-1, p67^phox and gp91^phox) in the subcutaneous adipose tissues of the stressed mice at 2 weeks of stress. A–D: Quantitative PCR data showing the levels of all targeted genes in both experimental groups at day 14 after stress. Results are mean ± SEM. *p<0.01 vs.

non-stressed group by one-way ANOVA followed by Tukey post hoc tests or Student's t-test.

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Suppl. Fig. S4. DPP-4 inhibition reduced the elevated numbers of blood platelets,

leukocytes (Leu), and neutrophils (Neu). A,B: Routine blood tests show the levels of blood platelets, leukocytes, and neutrophils in the stressed mice treated with vehicle or the DPP-4 inhibitor anagliptin (30 mg/kg twice daily) for 2 weeks. Results are mean ± SEM. *p<0.01 vs. non-stressed group by Student's t-test or one-way ANOVA followed by Tukey post hoc tests.

Suppl. Fig. S5. Anagliptin improved the alterations in the levels of the inflammatory- and oxidative stress-related genes (i.e., TNF-α, IL-1β, PAI-1, VCAM-1, ICAM-1, MCP-1, p67^phox and gp91^phox) in the subcutaneous adipose tissues of the stressed mice.

A–D: The PCR data showing the levels of all targeted genes in both experimental

groups at day 14 after stress. Results are mean ± SEM. *p<0.01 vs. non-stressed group by one-way ANOVA followed by Tukey post hoc tests or Student's t-test.

Suppl. Fig. S6. Anagliptin protected against harmful oxidative stress-induced changes

in cultured HUVECs. Following an overnight culture in serum-free medium, the HUVECs pretreated with anagliptin at the indicated concentrations were cultured in the presence or absence of H2O2 in serum-free EBM-2 for 24 hr and then subjected to a quantitative gene assay. A–E: The PCR data showing the levels of ADAMTS13 (A), eNOS (B), inflammatory genes (ICAM-1, VCAM-1, and MCP-1) (C,D), and proteolytic enzymes (CatS and CatK) (E) at the indicated concentrations of H2O2. The results are mean ± SEM. #p<0.01 vs. H2O2 at 0 μM, *p<0.01 vs. corresponding controls by one-way ANOVA followed by Tukey post hoc tests.

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Supplementary Table S1. Primer sequences for mice used for quantitative real-time PCR

Gene Forward Primer Reverse Primer

Mouse

p22phox AACTACCTGGAGCCAGTTGAG AATTAGGAGGTGGTGGAATATCGG gp91phox ACTTTCCATAAGATGGTAGCTTGG GCATTCACACACCACTCAACG p47phox CTGAGGGTGAAGCCATTGAGG GCCGGTGATATCCCCTTTCC

p67phox AACTACCTGGAGCCAGTTGAG AATTAGGAGGTGGTGGAATATCGG

MCP-1 GCCCCACTCACCTGCTGCTACT CCTGCTGCTGGTGATCCTCTTGT ICAM-1 CCCCGCAGGTCCAATTC CCAGAGCGGCAGAGCAA VCAM-1 ACAAAACGATCGCTCAAATCG GGTGACTCGCAGCCCGTA TNF-α AGGCTGCCCCGACTACGT GACTTTCTCCTGGTATGAGATAGCAAA

IL-1β TGCCACCTTTTGACAGTGATG ATGTGCTGCTGCGAGATTTG MMP-9 CCAGACGCTCTTCGA GAACC GTTATAGAAGTGGCGGTTGT

MMP-2 CCCCATGAAGCCTTGTTTACC TTGTAGGAGGTGCCCTGGAA TIMP-1 GCCTACACCCCAGTCATGGA GGCCCGTGATGAGAAACTCTT

TIMP-2 GTCCCATGATCCCTTGCTACA TGCCCATTGATGCTCTTCTCT eNOS TGTCTGCGGCGATGTCACT CATGCCGCCCTCTGTTG

ADAMTS13 GAGCGAATTCGCCGGCCACCATGAGCCAGCTTTGCCTGTG GAGCGGTACCGTGAGATACTAGTCTTCCCTGC

PAI-1 TCAGAGCAACAAGTTCAACTACACTGAG CCCACTGTCAAGGCTCCATCACTTGCCCA CatK AGCAGGCTGGAGGACTAAGGT TTTGTGCATCTCAGTGGAAGACT CatS GTGGCCACTA AAGGGCCTG ACCGCTTTTGTAGAAGAAGAAGGAG

IL-1β TGCCACCTTTTGACAGTGATG ATGTGCTGCTGCGAGATTTG GAPDH ATGTGTCCGTCGTGGATCTGA ATGCCTGCTTCACCACCTTCT Human

ICAM-1 CCTTCCTCACCGTGTACTGG AGCGTAGGGTAAGGTTCTTGC

VCAM-1 TGCACAGTGACTTGTGGACATA GCCACCACTCATCTCGATTT MCP-1 AGTCTCTGCCGCCCTTCT GTGACTGGGGCATTGATTG

ADAMTS13 GGACTCCCAGCAGGTATGG TGGGGGTAACTGTCAGAAATG

eNOS CACATGGCCTTGGACTGAA CAGAGCCCTGGCCTTTTC CatK ATATGTGGGACAGGAAGAGAGTTGT GGATCTCTCTGTACCCTCTGCATTTA

CatS AGGGAACTCATCAAAGACATCACTT GGGAATGCACTCATACGATCTG GAPDH GGACTTCGAGCAGGAGATGG GCACCGTGTTGGCGTAGAGG

ADAMTS13: a disintegrin and metalloproteinase with thrombospondin type 1 motif: member 13, CatK: cathepsin K, CatS: cathepsin S, eNOS: endothelial nitric oxide synthase, GAPDH:

gluceradehyde-3-phosphate dehydrogenase, ICAM-1: intercellular adhesion molecule-1, IL-1β:

interleukin-1β, MCP-1: monocyte chemoattractant protein-1, MMP-2: matrix metalloproteinase-2,

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MMP-9: matrix metalloproteinase-9, PAI-1: plasminogen activator inhibitor-1, TIMP-1: tissue inhibitor of matrix metalloproteinase-1, TNF-α: tumor-necrosis factor-α, VCAM-1: vascular cell adhesion molecule-1.

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