Nucleophosmin contributes to vascular inflammation and endothelial dysfunction in atherosclerosis progression

Journal Pre-proof Nucleophosmin contributes to vascular inflammation and endothelial dysfunction in atherosclerosis progression Caijun Rao, PhD, Baoqing Liu, PhD, Dandan Huang, PhD, Ru Chen, MD, Kai Huang, PhD, Fei Li, MD, Nianguo Dong, PhD PII:

S0022-5223(19)32776-X

DOI:

https://doi.org/10.1016/j.jtcvs.2019.10.152

Reference:

YMTC 15318

To appear in:

The Journal of Thoracic and Cardiovascular Surgery

Received Date: 7 June 2019 Revised Date:

15 October 2019

Accepted Date: 15 October 2019

Please cite this article as: Rao C, Liu B, Huang D, Chen R, Huang K, Li F, Dong N, Nucleophosmin contributes to vascular inflammation and endothelial dysfunction in atherosclerosis progression, The Journal of Thoracic and Cardiovascular Surgery (2020), doi: https://doi.org/10.1016/j.jtcvs.2019.10.152. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Copyright © 2019 Published by Elsevier Inc. on behalf of The American Association for Thoracic Surgery

Nucleophosmin contributes to vascular inflammation and endothelial dysfunction in atherosclerosis progression Caijun Rao，PhD1,2,3,a, Baoqing Liu, PhD4,a, Dandan Huang, PhD2,3,, Ru Chen, MD2,3, Kai Huang， PhD2,3,*, Fei Li, MD4,* , Nianguo Dong, PhD4 1

Department of Geriatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science

and Technology, Wuhan, China. 2

Clinical Center for Human Genomic Research, Union Hospital, Tongji Medical College, Huazhong

University of Science and Technology, Wuhan, China 3

Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science

and Technology, Wuhan, China 4

Department of Cardiovascular Surgery, Union Hospital, Tongji Medical College, Huazhong University

of Science and Technology, Wuhan, China a

Caijun Rao, Baoqing Liu contributed equally as first authors.

*Correspondence: Fei Li, MD, Department of Cardiovascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China, E-mail: [email protected] Co-Corresponding author: Kai Huang, FACC, PhD, Department of cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China, E-mail: [email protected]

Word account for the abstract: 248 Word account for the manuscript: 3665 Number of references: 35 Number of figures: 6 Number of supplemental figures/tables: 9 Conflict of interest: none declared. Funding: This study was supported by the National Natural Science Foundation of China (81670351, 81300173, 81570405 and 81670241). Central Picture Legend NPM accelerated the progression of atherosclerosis via NF-κB pathway. Central Message Nucleophosmin was enriched in human atherosclerotic plaque. Nucleophosmin aggravated atherosclerosis through promoting NF-κB pathway mediated vascular inflammation and endothelial dysfunction. Prospective Statement Nucleophosmin has been involved in the pathogenesis of many other diseases, however, its role in atherosclerosis remains unknown. This study provided novel evidence that nucleophosmin promoted atherosclerosis by inducing vascular inflammation and endothelial dysfunction through NF-κB signaling pathway, and suggested that nucleophosmin may be a promising target for atherosclerosis prevention and treatment. Video Legend This study uncovered a novel role of nucleophosmin in the progression of atherosclerosis. We found the level of nucleophosmin was higher in human atherosclerotic plaques than that in normal artery. Knockdown of nucleophosmin in vivo decreased macrophage infiltration, suppressed plaque formation and improved plaque stability in ApoE-/- mice. In vascular endothelial cells, nucleophosmin could bind with NF-κB p65, promote its intra-nuclear translocation and enhance the expression of

downstream target genes. Although this study has some limitations, we still believe that our findings are of general interest to the readers.

1

Nucleophosmin contributes to vascular inflammation and endothelial dysfunction in

2

atherosclerosis progression

3

Abstract

4

Objective: It was uncertain whether Nucleophosmin (NPM) participated in cardiovascular disease. The

5

present study aims to investigate the role and underlying mechanisms of NPM in atherosclerosis.

6

Methods： ：Levels and location of NPM in human carotid atherosclerotic plaques and healthy controls

7

were detected by Real-time PCR, immunoblots and immunofluorescence. Atherosclerotic prone ApoE-/-

8

mice were fed with western diet for 16 weeks as in vivo model. Human primary umbilical vein

9

endothelial cells (HUVECs) were cultured as in vitro model.

10

Results: Compared with controls, we found that NPM level in human carotid atherosclerotic plaques

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was more than twice as high as in normal arteries, which mainly localized in endothelial cells. In vivo,

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adenovirus containing NPM shRNA attenuated atherosclerotic lesion and promoted plaque stabilization

13

in western diet-fed ApoE-/- mice by reducing vascular inflammation, maintaining endothelial function

14

and decreasing macrophage infiltration. Furthermore, NPM knockdown decreased Nuclear factor-κB

15

(NF-κB) p65 phosphorylation. In cultured HUVECs, palmitic acid (PA) increased the protein levels of

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NPM, and induced the expression of inflammatory cytokines and monocyte adhesion, while NPM

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knockdown attenuated this effect. In HUVECs, NPM protein physically interacted with NF-κB p65

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subunit and promoted its nuclear transposition. NPM could also increase the transcriptional activity of

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NF-κB p65 promoter and enhance its binding to target genes including IL-1β, IL-6, ICAM-1 and

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E-selectin.

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Conclusions: These data provided novel evidence that NPM promoted atherosclerosis by inducing

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vascular inflammation and endothelial dysfunction through NF-κB signaling pathway, and suggested 1

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that NPM may be a promising target for atherosclerosis prevention and treatment.

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Keywords: Nucleophosmin; NF-κB; Vascular inflammation; Endothelial dysfunction; Atherosclerosis

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Introduction

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Atherosclerosis is a chronic vascular inflammatory disease which leads to some life-threatening

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complications, such as myocardial infarction and stroke. The increasing prevalence and mortality results

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in loss of productive life and death worldwide in recent years.

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risk factors is referred as a pathogenic sine qua non for atherosclerotic cardiovascular disease.

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Following endothelial impairment, circulating monocytes were then recruited to the intima and

31

differentiated to macrophages, which internalized modified lipoproteins to become foam cells (the

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hallmark of early lesions). 4

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1-3

Endothelial dysfunction induced by

It is noteworthy that Nuclear factor-κB (NF-κB) appears to play a pivotal role in the

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pro-inflammatory activation of endothelium in atherogenesis.

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transcription factor that can be activated by a variety of the pathophysiologic stimuli, resulting the

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regulation of multiple effector proteins associated with atherosclerosis such as inflammatory

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cytokines, chemokines, adhesion molecules and cell death signals. 7

5, 6

NF-κB is a pleiotropic

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Nucleophosmin (NPM, B23, Numatrin or NO38) is a widely expressed and critical

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phosphoprotein in the nucleoplasmin family. 8 NPM mainly exists in the nucleolus where it plays

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its major functions, while it can also convert back and forth between the nucleus and the

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cytoplasm.9, 10 NPM has been implicated in a number of pathways including ribosome biogenesis,

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chromatin remodeling, apoptosis and cell differentiation, participating several human

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malignancies.11, 12

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More recently, emerging evidence suggests that NPM controls DNA transcription through

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interaction with different transcription factors. NPM can either contribute to transcriptional activation,

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as in the report of Myc-interacting zinc finger protein 1 (Miz1)13, androgen receptor14, ribosomal 3

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genes15, c-Myc and Nuclear factor-κB (NF-κB)16, or to repression, as for the activating protein

48

transcription factor 2α (AP2α)17. These studies all point to the potentially vital function of NPM in

49

regulating gene expression in the transcription level.

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Nevertheless, little is known about the role and mechanisms of NPM in arterial pathophysiology.

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In this study, we intended to investigate the role and underlying mechanisms of NPM in exacerbating

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vascular inflammation and endothelial dysfunction in atherosclerosis progression

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4

54 55 56

Materials and Methods Expanded methods are available in supplemental data. Clinical Samples Collection

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All the studies involving humans complied with the declaration of Helsinki and were approved by

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the Ethics Committee of Huazhong University of Science and Technology. Written informed consent

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was obtained from all participants before surgery. Carotid atherosclerotic plaque specimens were

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explanted from patients undergoing carotid endarterectomy. Control specimens came from normal aorta

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of patients without atherosclerosis during aortic valve replacement.

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Animal Studies

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All animal studies followed the guidelines of the Animal Care and Use Committee of Huazhong

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University of Science and Technology. Eight-week-old male ApoE-/- mice were randomly devided into

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4 groups: ApoE-/- mice fed with a chow diet or western diet for 16 weeks treated with Ad-shNC or

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Ad-shNPM respectively (n=18 for each group). Four weeks before sacrificed, mice were injected with

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5×108 plaque-forming unites (5×108 pfu/mouse) either Ad-shNC or Ad-shNPM adenovirus via the tail

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vein every two weeks. Mice were housed individually under a 12h light/12h dark cycle and sacrificed

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by cervical dislocation.

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NPM Knockdown in Vivo and in Vitro

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Adenovirus containing specifically designed shRNA was used to knock down NPM expression in

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animal experiments. siRNA was transfected into HUVECs to knockdown NPM expression in cellular

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experiments..

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Cell Culture and Treatment

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Human primary umbilical vein endothelial cells (HUVECs) were isolated from umbilical veins by 5

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using 0.1% collagenase I (Sigma-Aldrich) and cultured in specific endothelial cell medium (ECM)

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(Sciencell) with 10% fetal bovine serum (FBS) according to the reagent manufacturer's instructions.

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HUVECs between passages 3 to 8 were used and treated with palmitic acid (PA) as in vitro model.

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Adhesion Assay

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Adhesion assay was performed using Endothelial Cell Adhesion Assay Kit (ECM645; Bioscience

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Research Reagents) according to the manufacturer's instructions.

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Immunoprecipitation Assay

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Immunoprecipitation assay (IP) was carried out as described in previous studies. 500 µg of total

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proteins were incubated with NF-κB p65 antibody (8242S; CST) and NPM antibody (ab10530; Abcam)

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at 4 degree overnight with protein A+G magnetic beads (Millipore). The immunoprecipitates were

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washed and centrifuged 4 times with lysis buffer, following by western blot analysis. Immunoglobulin

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G (IgG) from the same species was used as negative control.

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Gene Overexpression

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Human NPM plasmid was obtained from ORIGENE (RC203841), full length of human NPM

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coding sequence was constructed in a functional mammalian expression vector pCMV6. pCMV6-Entry

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vector was used as a negative control (Entry vector). The cultured HEK293 cells and HUVECs were

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transfected with plasmids using lipofectamine 2000 (Invitrogen) for 24 hours before subsequent

93

experiments.

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Luciferase Assay

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By using the One Step Cloning Kit (C112-02; Vazyme Biotech), NF-κB luciferase reporter plasmid

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was purchased from Tsingke Biotechnology, promoters of ICAM-1, VCAM-1, E-selectin, and P-selectin

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were assembled into pGL3.0 vector (Promega). Primers used above were listed in Supplemental Table 3. 6

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HEK293 cells were transfected with an internal control plasmid (pRL-TK; Promega) and plasmids

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containing other promoter regions simultaneously. Luciferase activity were detected with the Dual

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Luciferase Reporter Assay Kit (Promega).

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Chromatin Immunoprecipitation Assay

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Chromatin immunoprecipitation assay (ChIP) was performed by ChIP assay kit (Millipore)

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according to the manufacturer’s instructions. Using PCR to detect DNA samples enriched by NF-κB

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p65 antibody (8242S; CST) to test whether the downstream target genes have changed. Rabbit IgG

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antibody was performed as negative control. Primers used in ChIP assay were shown in Supplemental

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Table 4.

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Statistical Analysis

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For the data of human characteristics, continuous variables were shown as mean ± SD when it was

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normally distributed or median (interquartile range) when it was not and evaluated by Student’s t test or

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nonparametric test when appropriate, categorical variables were presented as percentages and compared

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between groups with chi-square tests.18

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For the results of animal and cellular experiments, data were presented as the mean ± SD,

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statistical differences were evaluated by unpaired Student’s t-test between two groups or one-way

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analysis of variance (ANOVA) for multiple group comparison. Bonferonni corrections were made

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for the multiple testing. All analysis was performed with SPSS 23.0, P<0.05 was characterized as

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statistically significant.

7

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Results

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NPM was enriched in human atherosclerotic plaque

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To explore the involvement of NPM in atherosclerosis, we first examined the level of NPM in 12

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carotid atherosclerotic plaques and 12 human healthy arteries. The demographic characteristics of

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patients were summarized in Supplemental Table 1. The level of NPM mRNA was more than twice as

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high as that in normal arteries, immunoblots also indicated that the protein level of NPM was elevated

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in plaque tissues (Figure 1A and 1B). To determine the cellular localization of NPM, we performed dual

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immunofluorescent staining of human carotid atherosclerotic plaques using antibodies against NPM and

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CD31 (marker of endothelial cells), CD68 (marker of macrophages) or α-SMA (marker of smooth

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muscle cells). As shown in Figure 1C, NPM predominantly co-localized with CD31 positive cells,

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suggesting that NPM was mainly expressed in VECs.

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Inhibition of NPM attenuated endothelial dysfunction and atherosclerosis in ApoE-/-mice

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To evaluate the effect of NPM on atherosclerosis in vivo, we generated recombinant adenovirus

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containing NPM shRNA (shNPM) and injected it into ApoE-/- mice via tail vein. As confirmed in Figure

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2A and 2B, Ad-shNPM inhibited the transcriptional and translational expression of NPM by 65.2% in

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the murine aorta. Oil Red O staining showed that Ad-shNPM decreased lesion area at aortic sinus by

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45.1% compared to the control treated with Ad-shNC in western diet-fed ApoE-/- mice, while none

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significant beneficial effect was observed in mice fed with chow diet (Figure 2C). Likewise, NPM

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knockdown markedly reduced atherosclerotic plaque formation (decreased by 51.3% compared to

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Ad-shNC-treated mice, Figure 2D). Knockdown of NPM also improved atherosclerotic plaque stability,

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as shown in Figure 2E, Ad-shNPM-treated mice exhibited smaller necrotic core area, thicker fibrous

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caps, less elastic fiber breaks, as well as decreased macrophage infiltration. Furthermore, Ad-shNPM 8

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suppressed NF-κB p65 phosphorylation and decreased the protein level of VCAM-1, ICAM-1,

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E-selectin, indicating that knockdown of NPM attenuated endothelial dysfunction and inflammation in

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western diet-fed ApoE-/- mice (Figure 2F).

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Knockdown of NPM suppressed the inflammatory responses in HUVECs

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To investigate the role of NPM in endothelial function, we knocked down NPM in HUVECs by

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siRNA. RT-PCR and Western blot showed that NPM siRNA efficiently decreased the expression of

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NPM (Supplemental Figure 3A and 3B). To explore whether NPM was involved in the regulation of

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endothelial cell inflammatory reactions, we tested mRNA levels of cytokines and adhesion molecules in

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HUVECs stimulated by palmitic acid (PA)19 with or without NPM knockdown (Figure 3A). Depletion

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of NPM with siRNA notably reduced the levels of IL-1β, IL-6 and TNF-α in the supernatants of

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HUVECs incubated with PA (Figure 3B). We further characterized the effect of NPM on the expression

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adhesion molecules. We measured the expression of ICAM-1, VCAM-1, and E-selectin by western blot

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analysis, and showed that knockdown of NPM suppressed the expression of all these molecules in

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varying degrees (Figure 3C). After that, we generated endothelial cell monolayer adhesion assay using

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immunofluorescence technique. Consistent with the above results, NPM knockdown could significantly

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reduce the number of peripheral blood monocytes attached to PA-activated endothelial cell monolayers

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(Figure 3D).

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NPM physically interacted with NF-κ κB p65 subunit and promoted its activation

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To uncover the underlying mechanism of the above observations, we explored whether NF-κB

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pathway, a crucial pro-inflammatory transcription factor, was involved in the process. The

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phosphorylation and nuclear translocation of NF-κB p65 subunit is indispensable for its transcriptional

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activation, we measured the expression level of phospho-p65 (p-p65) to evaluate the activation status of 9

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NF-κB not only in total protein but also in nuclear extract and cytoplasmic extract respectively (Figure

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4A and 4B). Immunofluorescent staining showed that PA promoted nucleus translocation of p65, which

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could be prevented by NPM siRNA (Figure 4C). To investigate how NPM modulated NF-κB

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transcriptional activity, we hypothesized that NPM could function as a transcriptional co-activator that

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interacted with NF-κB via physical binding. We immunoprecipitated NF-κB p65 in untreated cells, and

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detect the bound form of NPM with western blot. Conversely, immunoprecipitation of NPM resulted in

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the co-precipitation of NF-κB p65. Furthermore, treatment with PA strengthened their interaction with

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each other in HUVECs (Figure 4D). PA treatment and NPM over-expression by transfecting NPM

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plasmid significantly augmented the promoter activity of NF-κB p65, which could also be inhibited by

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NPM siRNA or NF-κB inhibitor PDTC20-22 (Figure 4E).

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NPM enhanced the transcriptional activity of NF-κ κB to its downstream target genes

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NF-κB regulates gene expression through binding to specific sites in the promoter of target genes.

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We examined the promoter activities of the adhesion molecules includingICAM-1, VCAM-1, E-selectin,

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and P-selectin. PA treatment and NPM over-expression significantly up-regulated the luciferase

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activities of all promoters except for ICAM-1, whereas the increased luciferase activity could be

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reduced by NF-κB inhibitor PDTC (Figure 5A). To further determine the influence of NPM on the DNA

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binding activity of NF-κB, chromatin immunoprecipitation (ChIP) experiments was performed. NPM

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over-expression could enhance more NF-κB binding to its downstream gene promoters, including IL-1β,

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IL-6, ICAM-1 and E-selectin (Figure 5B).

10

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Discussion

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Currently, little is known about NPM involvement in the progression of atherosclerosis. The

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present study showed that NPM was enriched in human atherosclerotic plaque and depletion of NPM

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attenuated the atherosclerotic lesions in ApoE-/- mice fed with western diet. This effect was

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accompanied by reduced NF-κB activation as well as macrophage infiltration and adhesion molecules

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expression. In vitro studies showed that NPM knockdown significantly inhibited inflammatory

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cytokines production (including IL-1, IL-6 and TNF-α) and adhesion molecules (including ICAM-1,

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VCAM-1 and E-selectin) as well as NF-κB p65 intranuclear translocation in cultured HUVECs

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stimulated by PA. Furthermore, NPM modulated NF-κB transcriptional activity via physical interaction

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with p65 subunit. To our knowledge, the novel findings provided the first evidence that

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pro-inflammatory effects induced by NPM/NF-κB interaction in VECs contributed to the development

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and progression of atherosclerosis.

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NPM is an important nucleocytoplasmic shuttling protein which has been reported to participate in

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metabolic pathways, DNA repair pathways and regulating apoptosis. NPM malfunction such as

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overexpressed, mutated, rearranged or deleted has been described in a varieties of cancers including

195

lymphoma, acute myeloid leukemia, breast cancer and gastric cancers.23 However, emerging evidence

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suggests that NPM-mediated nucleolar stress is involved in cardiovascular disease such as myocardial

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infarction and hypertension.24, 25 tudies also linked NPM to the onset of atherosclerosis. NPM mRNA

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expression was increased in carotid arteries of aged rats.26 Oxidized low density lipoprotein decreased

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human VECs proliferation through NPM dephosphorylation.27 Consistent with previous studies, our

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findings further provided a more robust evidence that NPM mRNA and protein levels were increased in

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human atherosclerotic plaques and mainly localized in vascular endothelial cells. In addition, 11

202

knockdown of NPM in vivo attenuated atherosclerotic lesions in western diet-fed ApoE-/- mice.

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Endothelial cells not only function as a barrier but also can undergo a dramatic transition in their

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phenotype in response to injury or other biological stimuli including pathogen-associated molecular

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patterns (PAMPs) and other damage-associated

206

pro-inflammatory activation resulted in the expression of leukocyte adhesion molecules, such as

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ICAM-1, VCAM-1 and E-selectin, which selectively recruited circulating monocytes into intima, where

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they differentiated into macrophages and internalize modified lipoproteins to become foam cells. In our

209

study, NPM knockdown inhibited the production of inflammatory cytokines and adhesion molecules in

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HUVECs, thus significantly reduced attachment of monocytes to endothelial cells. In addition,

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knockdown of NPM in vivo also resulted in decreased expression of ICAM-1, VCAM-1 and E-selectin

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as well as reduced macrophage infiltration.

molecular patterns (DAMPs). Endothelial

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Highly unstable plaques are thought to be fast-growing and accompanied with superimposed

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thrombosis, eventually leading to acute adverse cardiovascular events.28 Typical features of the

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vulnerable plaques include massive infiltration of inflammatory cells, and a large necrotic core with thin

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fibrous cap.29 Compared with control, Ad-shNPM treated mice turned out to have thicker fibrous caps,

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smaller central necrotic areas and less elastic fiber breaks, suggesting that in addition to the reduction in

218

lesion size, the plaque stability was also greatly improved. From the above, NPM overexpression

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probably aggravated atherosclerotic lesion and plaque unstability through inducing endothelial

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pro-inflammation change. However, these indicators have their limitation and can only indirectly reflect

221

plaque stability.

222

more properly.

223

30-32

Further studies are needed to investigate the effect of NPM on plaque stability

Nuclear factor-κB (NF-κB) mediated pro-inflammatory signaling pathway plays a crucial role in 12

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endothelial dysfunction. Our findings demonstrated knockdown of NPM inhibited PA-induced NF-κB

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p65 subunit phosphorylation and intra-nuclear translocation. Furthermore, we found physical interaction

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between NPM and NF-κB p65 subunit. NPM also enhanced p65 promoter activity. Pretreatment with

227

NF-κB inhibitor decreased adhesion molecules promoter activity induced by NPM over expression. All

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these results demonstrated that NF-κB activation might be responsible for the detrimental role of NPM

229

in atherosclerosis. Interestingly, recent studies suggested that NPM level tended to be upregulated

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during aging heart, and SIRT1 may prevent NPM-induced NF-κB activation.

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warranted to clarify the role of NPM in regulation of pathophysiological conditions associated with

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arterial aging.

26

Further studies are

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In summary, the present study uncovered a previously unrecognized role of NPM in exacerbating

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endothelial inflammation during the progression of atherosclerosis in ApoE-/- mice. NPM knockdown

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abrogated PA-induced expression of inflammatory cytokines as well as adhesion molecules in cultured

236

HUVECs and reduced atherosclerosis plaque formation in western diet-fed ApoE

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protective effects were probably mediated via inhibition of NF-κB signaling pathway (Figure 6). All the

238

above-mentioned suggested NPM may be a promising target for slowering atherosclerosis, stabilizing

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plaque and reducing associated adverse events.

240

Study limitations

-/-

mice. These

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Some limitations existed in our work. One was that we did not examine the effect of NPM

242

knockdown on the metabolism of serum lipids in ApoE -/- mice. The other was that we performed all the

243

works on male mice, whether NPM made sense in female animals remained unknown, for male and

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female differed in the development of atherosclerosis.33,

245

(VSMCs) can also express ICAM-1 and VCAM-1 in atherosclerosis35, whether NPM can directly 13

34

Besides, vascular smooth muscle cells

246

regulate SMC function is also unknown. Furthermore, although the specimens were ample enough to

247

study, the result would be more robust if increasing the sample size for some comparison.

248

Conflict of interest: none declared.

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Funding: This study was supported by the National Natural Science Foundation of China (81670351,

250

81300173, 81570405 and 81670241).

14

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Figure legends

355

Figure 1. NPM was enriched in human atherosclerotic plaques. (A) Quantitative Real-time

356

PCR showed that NPM mRNA expression was markedly increased in human carotid

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atherosclerotic plaques compared with healthy arteries, n=12, **P ＜ 0.01 vs normal. (B)

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Immunoblots showed that NPM protein level in atherosclerotic plaques was significantly higher

359

than that in control, n=6, ***P ＜ 0.001 vs normal. (C) Representative images of dual

360

immunofluorescent staining showed that NPM was predominantly expressed in endothelial cells.

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For co-localization analysis, sections were dual-stained for NPM (green) and CD31 (red,

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endothelial marker), a-SMA (red, smooth muscle cell marker), or CD68 (red, macrophage marker).

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DAPI was used for nucleus staining (blue). NPM and endothelial cell-specific marker

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double-positive cells were indicated by arrows.

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Figure 2. Inhibition of NPM attenuated endothelial dysfunction and atherosclerosis in

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western diet-fed ApoE-/-mice. (A and B) Recombinant adenovirus (Ad-shNPM) significantly

367

decreased the protein and mRNA level of NPM in the murine vessel, n=3, **P＜0.01 vs shNC. (C)

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ApoE-/- mice fed a chow diet (upper) or a western diet (lower) were injected with Ad-shNC or

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Ad-shNPM. Oil Red O staining showed that Ad-shNPM decreased lesion area at aortic sinus in

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western diet-fed ApoE-/- mice, n=6, **P＜0.01 vs shNC. (D) Representative images of Oil Red O

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staining in the whole aorta. Quantification analysis showed that Ad-shNPM markedly reduced

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atherosclerotic plaque formation in western diet-fed ApoE-/- mice, n=6, ***P＜0.001 vs shNC. (E)

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Representative images of aortic sinus sections stained with Hematoxylin-eosin (HE), Masson’s

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trichrome (Masson), elastica van Gieson (EVG) and immunohistochemical staining for α-SMA

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and CD68. Circular regions mark the necrotic core area. The black arrows indicate α-SMA 20

376

staining panels where fibrous caps were formed, which can be used for calculating the thickness of

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the fibrous caps. The white arrows in EVG staining panels indicate the rupture of elastic fibers of

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tunica media. Histological quantification analysis showed that Ad-shNPM changed the

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pathological features of atherosclerotic lesions compared with Ad-shNC, n=6, *P＜0.05, **P＜

380

0.01, ***P＜0.001 vs shNC. (F) Western blot was performed to detect the protein levels of

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ICAM-1, VCAM-1, E-selectin, total and phosphorylated NF-κB p65 subunit in the vessel tissues

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of ApoE-/- mice. Densitometric analysis showed that Ad-shNPM suppressed NF-κB p65

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phosphorylation and decreased the protein level of VCAM-1, ICAM-1, E-selectin, n=6, *P＜0.05,

384

**P＜0.01 vs shNC. Data are expressed as mean ± SD, “n. s.” means no statistical significance.

385

Figure 3. Knockdown of NPM suppressed the inflammatory responses in HUVECs. (A)

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Real-time PCR showed that knockdown of NPM by siRNA decreased mRNA expression levels of

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inflammatory chemokines, adhesion molecules and procoagulant factors in HUVECs treated with

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500 µmol/L palmitic acid (PA) for 24 h, n=3, *P＜0.05, **P＜0.01 vs scramble siRNA. (B)

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Knockdown of NPM with siRNA notably reduced the levels of IL-1β, IL-6 and TNFα in the

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supernatants of HUVECs treated with 500 µmol/L PA for 24 h, n=5, *P＜0.05, **P＜0.01, ***P

391

＜0.001 vs scramble siRNA. (C) Immunoblot for ICAM-1, VCAM-1, E-selectin and P-selectin in

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HUVECs showed that NPM knockdown suppressed the expression of all these adhesion

393

molecules, n=3, *P＜0.05 vs scramble siRNA. (D) NPM knockdown significantly reduced the

394

number of peripheral blood monocytes attached to PA-activated endothelial cell monolayers in the

395

adhesion assay, n=3, *P＜0.05, **P＜0.01 vs scramble siRNA. Data are expressed as mean ± SD,

396

“n. s.” means no statistical significance.

397

Figure 4. NPM physically interacted with NF-κ κB p65 subunit and promoted its activation. (A) 21

398

Immunoblots analysis of p65, Iκbα and their phosphorylated protein level in total HUVECs

399

protein after stimulated with 500 µmol/L PA for 1h, and NPM siRNA significantly decreased the

400

ratio of p-p65/p65 and p-Iκbα/Iκbα, n=3, *P＜0.05 vs scramble siRNA. (B) Immunoblots of p65

401

protein level in nuclear extract and cytoplasmic extract showed that PA increased and decreased

402

the expression of p65 in nucleus as well as in cytoplasm respectively, which were both inhibited

403

by NPM siRNA. n=3, *P＜0.05 vs scramble siRNA. (C) Representative immunofluorescent

404

staining against p65 (green) showed that NPM knockdown inhibited p65 intranuclear translocation

405

induced by PA. The nucleus was stained with DAPI (blue). (D) Immunoblots for NPM and NF-κB

406

p65 after immunoprecipitation with anti-p65 antibody, anti-NPM antibody demonstrated physical

407

binding of NPM and NF-κB. PA stimulation strengthened their interaction with each other in

408

HUVECs. (E) Luciferase assay was used to measure NF-κB promoter activity. Luciferase reporter

409

construct containing the promoter of p65 was transfected with an internal control plasmid pRL-TK

410

into HEK293 cells. PA treatment and NPM overexpression significantly augmented the promoter

411

activity of NF-κB p65, which could be inhibited by either NPM siRNA or NF-κB inhibitor PDTC,

412

n=3, **P＜0.01. Data are expressed as mean ± SD, “n. s.” means no statistical significance.

413

Figure 5. NPM enhanced the transcriptional activity of NF-κ κB to its downstream target

414

genes. (A) Luciferase reporter constructs containing promoters of ICAM-1, VCAM-1, E-selectin

415

and P-selectin were co-transfected with an internal control plasmid pRL-TK into HEK293 cells.

416

PA treatment and NPM overexpression significantly augmented the promoter activity of these

417

adhesion molecules (excluding ICAM-1), which could also be inhibited by NPM siRNA or NF-κB

418

inhibitor PDTC, n=3, **P＜0.01, ***P＜0.001. (B) Chromatin immunoprecipitation (ChIP) assay

419

was performed in cultured HUVECs using ChIP standard p65 antibody. IgG acted as negative 22

420

control. Percentage of amplified signals from IP chromatin to amplified input signals obtained

421

from the same sample indicated that NPM overexpression could significantly enhance NF-κB p65

422

binding to its downstream gene promoters, including IL-1β, IL-6, ICAM-1 and E-selectin, n=3, *P

423

＜0.05, **P＜0.01 vs IgG group. Data are expressed as mean ± SD, “n. s.” means no statistical

424

significance.

425

Figure 6. Graphical abstract of how nucleophospmin contributed the progression of

426

atherosclerosis. Risk factors induced the expression of nucleophosmin, which subsequently increased

427

the phosphorylation of NF-κB p65 subunit and promoted its translocation to nucleus, resulting

428

production of inflammatory cytokines and adhesion molecules. Vascular inflammation and endothelial

429

dysfunction aggravated atherosclerosis progression.

23