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Klf5 down-regulation induces vascular senescence through eIF5a depletion and mitochondrial fission

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Open Access
Peer-reviewed

Research Article

Dong Ma, 

Bin Zheng, 

He-liang Liu, 

Yong-bo Zhao, 

Xiao Liu, 

Xin-hua Zhang, 

Qiang Li, 

Wei-bo Shi, 

Toru Suzuki, 

Jin-kun Wen

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Published: August 20, 2020

https://doi.org/10.1371/journal.pbio.3000808

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AbstractAlthough dysregulation of mitochondrial dynamics has been linked to cellular senescence, which contributes to advanced age-related disorders, it is unclear how Krüppel-like factor 5 (Klf5), an essential transcriptional factor of cardiovascular remodeling, mediates the link between mitochondrial dynamics and vascular smooth muscle cell (VSMC) senescence. Here, we show that Klf5 down-regulation in VSMCs is correlated with rupture of abdominal aortic aneurysm (AAA), an age-related vascular disease. Mice lacking Klf5 in VSMCs exacerbate vascular senescence and progression of angiotensin II (Ang II)–induced AAA by facilitating reactive oxygen species (ROS) formation. Klf5 knockdown enhances, while Klf5 overexpression suppresses mitochondrial fission. Mechanistically, Klf5 activates eukaryotic translation initiation factor 5a (eIF5a) transcription through binding to the promoter of eIF5a, which in turn preserves mitochondrial integrity by interacting with mitofusin 1 (Mfn1). Accordingly, decreased expression of eIF5a elicited by Klf5 down-regulation leads to mitochondrial fission and excessive ROS production. Inhibition of mitochondrial fission decreases ROS production and VSMC senescence. Our studies provide a potential therapeutic target for age-related vascular disorders.

Citation: Ma D, Zheng B, Liu H-l, Zhao Y-b, Liu X, Zhang X-h, et al. (2020) Klf5 down-regulation induces vascular senescence through eIF5a depletion and mitochondrial fission. PLoS Biol 18(8):
e3000808.

https://doi.org/10.1371/journal.pbio.3000808Academic Editor: Cecilia W. Lo, University of Pittsburgh, UNITED STATESReceived: December 25, 2019; Accepted: July 31, 2020; Published: August 20, 2020Copyright: © 2020 Ma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Data Availability: All relevant data are within the paper and its Supporting Information files. All data of microarray and RNA sequencing used in this paper are from NCBI, and the accession numbers from NCBI are GSE148841 and GSE148765, respectively. Data used for figure generation are provided in the Figures.data and supplemental Figures.data Excel sheets. All images of blot and gel are available from the PDF file.Funding: J-KW received funding from the National Natural Science Foundation of China (No. 31671182 and No. 31871152) and Hebei scientific research project of high level talents (GCC2014026). DM is funded by the National Natural Science Foundation of China (No. 81700416). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing interests: The authors have declared that no competing interests exist.Abbreviations:
AAA,
abdominal aortic aneurysm; Ad-Ctl,
adenoviruses encoding control; Ad-Klf5,
adenoviruses encoding Klf5; Ad-shKlf5,
adenoviruses encoding small hairpin Klf5; Ad-U6,
adenoviruses encoding U6; Ang II,
angiotensin II; Atp5b,
ATP synthase subunit β; Cdkn1a,
cyclin dependent kinase inhibitor 1a; Cox4i1,
cytochrome c oxidase subunit 4i1; Cox6a2,
mitochondrial cytochrome c oxidase subunit 6A isoform 2; CT,
computed tomography; DHE,
dihydroethidium; DMEM,
Dulbecco’s Modified Eagle Medium; Drp1,
dynamin-related protein 1; EC,
endothelial cell; eIF5a,
eukaryotic translation initiation factor 5a; ERK,
extracellular signal–regulated kinase; FACS,
flow analysis of cytosorting; Fis1,
fission mitochondrial 1; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; GO,
gene ontology; GPCR,
G protein–coupled receptor; HE,
hematoxylin–eosin; IgG,
immunoglobulin G; KEGG,
Kyoto Encyclopedia of Genes and Genomes; Klf5,
Krüppel-like factor 5; Mapk14,
mitogen-activated protein kinase 14; MCP-1,
monocyte chemoattractant protein 1; Mdivi-1,
mitochondrial division inhibitor 1; Mfn1,
mitofusin 1; Mfn2,
mitofusin 2; MMP2,
matrix metalloproteinase 2; mtDNA,
mitochondrial DNA; Mtfr1,
mitochondrial fission regulator 1; MTG,
MitoTracker Green; mtROS,
mitochondrial ROS; mtTFA,
mitochondrial transcription factor A; NAC,
N-acetyl-L-cysteine; Nfe2l2,
nuclear factor, erythroid 2 like 2; NOX,
NAPDH oxidase; PDGF,
platelet-derived growth factor; PGC1α,
peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; Pink1,
PTEN-induced kinase 1; PPARα,
peroxisome proliferator-activated receptor α; pRL-TK,
thymidine kinase promoter-Renilla luciferase reporter plasmid; qRT-PCR,
quantitative real-time PCR; RNA-Seq,
RNA sequencing; ROS,
reactive oxygen species; SA-β-gal,
senescence-associated β-galactosidase; Si-Ctl,
short interfering RNA control; Si-Drp1,
short interfering RNA targeting Drp1; Si-eIF5a,
short interfering RNA targeting eIF5a; Si-Mfn1,
short interfering RNA targeting Mfn1; siRNA,
small interfering RNA; SMA,
smooth muscle α-actin; Tmx2,
thioredoxin-related transmembrane protein 2; VEGF,
vascular endothelial growth factor; VSMC,
vascular smooth muscle cell; XPO1,
nuclear export protein exportin 1; WT,
wild-type; ΔΨm,
mitochondrial membrane potential

IntroductionCellular senescence is an important contributor to aging and age-related diseases, and the accumulation of cellular senescence is a main feature of aged organisms [1]. Cellular senescence is traditionally defined as permanent cell cycle arrest in response to different damaging stimuli [1,2]. In the cardiovascular system, the senescence of vascular smooth muscle cells (VSMCs) may be induced by different stimuli, such as angiotensin II (Ang II), oxidative stress, inflammation, and DNA damage. On the other hand, VSMC senescence may also results in the loss of arterial function, chronic vascular inflammation, mitochondrial dysfunction, and the development of age-related vascular disorders, such as atherosclerosis, abdominal aortic aneurysm (AAA), hypertension, and diabetes [3,4].
Krüppel-like factor 5 (Klf5) is a zinc-finger transcriptional factor that regulates various cellular processes, including proliferation, differentiation, development, and apoptosis [5]. In VSMCs, Klf5 is regulated by Ang II signaling and is an essential regulator of cardiovascular remodeling [6]. Ang II is known not only to regulate blood pressure and electrolyte balance but also to be involved in mediation of cell proliferation and oxidative stress, thus contributing to premature senescence [7,8]. During cardiovascular remodeling, Klf5 activates the expression of cell cycle promoting genes, such as cyclin D1, cyclin B1, and growth factors and their receptors, such as platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and VEGF receptor [9,10], with a concomitant suppression of negative cell cycle control gene p21 [11,12]. Our recent studies demonstrate that Klf5 is highly expressed in both macrophages and VSMCs of human and experimental mouse AAA. Moreover, Klf5 expression progressively increases with the aortic diameter expansion during Ang II infusion–induced AAA formation [13]. Despite the facts that (1) advanced age is a major risk factor for AAAs [14], (2) VSMC senescence correlates with increased level of reactive oxygen species (ROS) [15], and (3) Klf5 participates in development and progression of AAAs [13], it remains to be clarified whether and how Klf5 mediates the mechanistic and functional link between ROS generation and VSMC senescence.
Mitochondria play important roles in regulating critical cellular, physiological, and pathophysiological processes [16,17]. Alterations in mitochondrial dynamics, which is regulated mainly by the processes of fission and fusion, are implicated in various human diseases, including cancer and neurologic and cardiovascular diseases [17–19]. Previous studies have demonstrated that dysregulation of mitochondrial dynamics is a key feature of aging [17]. Moreover, mitochondria are a major source of ROS, and mitochondrial dysfunction may lead to aberrant ROS production [20]. Specifically, elevated ROS levels impair vascular cell life span through the onset of cellular senescence [7] and have been demonstrated in human cerebral aneurysm [21]. These results indicate that dysregulation of mitochondrial dynamics can drive VSMC senescence and excessive ROS production. Although cardiomyocyte Klf5 was recently identified as a regulator of cardiac metabolism by directly activating transcription of peroxisome proliferator-activated receptor α (PPARα) and regulating lipid metabolism [22], whether and how Klf5 regulates mitochondrial dynamics is currently unclear.
It is well known that the dynamin-like GTPases mitofusin 1 and 2 (Mfn1, Mfn2) regulate the mitochondrial fusion process, and dynamin-related protein 1 (Drp1) plays a central role in the regulation of mitochondrial fission [23]. Eukaryotic translation initiation factor 5a (eIF5a) is localized not only to the nucleus but also to the mitochondria [24] and is involved in the regulation of redox homeostasis [25]. However, it remains unclear whether eIF5a, together with mitochondrial dynamics–related proteins, participates in the regulation of mitochondrial dynamics. Therefore, it is very intriguing to investigate whether eIF5a mediates functional link between Klf5 and mitochondrial dynamics, as well as to explore the relationship between Klf5-regulated mitochondrial dynamics and VSMC senescence.

Results
Down-regulation of Klf5 expression in VSMCs is correlated with the progression and rupture of aortic aneurysm
Because Klf5 is well known to mediate Ang II–induced vascular remodeling by stimulating VSMC proliferation [5], we sought to know how medial VSMCs were lost in Ang II–induced AAA. Thus, we determined the expression of Klf5 in human unruptured and ruptured AAAs. Demographic characteristics and representative three-dimensional volume-rendered images from axial computed tomography (CT) scans as well as histological analyses with hematoxylin–eosin (HE) and Masson’s trichrome staining are presented in Table 1 and S1A–S1C Fig. And the results of western blot showed that Klf5 expression in ruptured AAA (4 cases) was significantly lower than that in unruptured AAA (22 cases), even though its expression was higher than that in normal abdominal aorta (8 cases) (Fig 1A and 1B). Furthermore, confocal microscopy images were obtained by immunofluorescence staining with VSMC marker (smooth muscle α-actin, SMA) and Klf5, and showed that Klf5 was co-localized with VSMC marker in unruptured AAA (Fig 1C). Moreover, Klf5-positive VSMCs were hardly observed in ruptured AAA, while the percentages of Klf5-positive VSMCs were significantly higher in unruptured AAA than in the normal aorta (10.3% ± 2.2% versus 2.4% ± 0.89%; Fig 1C and 1D).
Fig 1. Klf5 expression is down-regulated in the ruptured human and experimental AAA.(A) The images of all western blots of Klf5 in human unruptured (n=22) and ruptured (n=4) AAA samples and in control aortic tissues (n=8). (B) Band intensities that were measured and normalized to β-actin. *P 30% increase compared to young WT mice (1.73 ± 0.31 mm versus 1.25 ± 0.28 mm, P 30% increase in the aortic diameter compared with WT mice (2.05 ± 0.38 mm versus 1.71 ± 0.33 mm, P Read More

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