KAP1 silencing relieves OxLDL-induced vascular endothelial dysfunction by down-regulating LOX-1

Atherosclerosis (AS) is a multifactorial disease, and the cellular and molecular mechanisms underlying AS are unclear. An increasing number of studies have confirmed that atherosclerosis is a chronic inflammatory disease caused by endothelial dysfunction (ED) [1,2]. ED is widely accepted to be an early sign of AS, and oxidized low-density lipoprotein (OxLDL) is thought to be an initial risk factor for ED [3]. Analyses from clinical data have revealed that elevated levels of OxLDL and other oxides in plasma are associated with vascular ED [4,5]. In response to various types of stress, endothelial cells (ECs) may change their functions and initiate the synthesis and release of vasoactive factors, cytokines, or growth factors. These alterations, referred to as ED, contribute to atherosclerotic plaque formation, lesion progression, and ultimately plaque rupture [6].

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), a key scavenger receptor, mediates the binding of OxLDL in vascular cells and induces the activation of signaling pathways involved in the development of oxidative stress and inflammation [7]. LOX-1 mediates OxLDL-induced production of reactive oxygen species (ROS) and adhesion molecules in atherosclerosis [8]. In addition, LOX-1 plays a pivotal role in OxLDL-mediated AS via endothelial nitric oxide synthase (eNOS) uncoupling and nitric oxide (NO) reduction. NO derived from eNOS plays a crucial role in maintaining endothelial homeostasis [9]. ECs incubated with OxLDL (12–24 h) have been shown to induce ED by up-regulating the expression of LOX-1 and inhibiting the function of eNOS [10]. Therefore, the OxLDL/LOX-1 pathway has been implicated in the pathogenesis of ED.

KRAB domain-associated protein 1 (KAP1), also called Triple motif protein 28 (TRIM28) or transcriptional intermediary factor 1 beta (TIF1β), is an essential cofactor of Kruppel-associated box zinc finger proteins (KRAB-ZFPs). KAP1 serves as a transcriptional co-repressor that regulates the transcription of KRAB-ZFP-specific loci by binding to the conserved KRAB domain. Despite the formation of complexes with KRAB-ZFPs, KAP1 also regulates the activity of transcription factors lacking the KRAB domain, such as c-Myc and E2F. KAP1 can also epigenetically modulate chromatin structure by recruiting histone deacetylase, histone methyltransferase, and heterochromatin protein 1 [11]. KAP1 silencing has been shown to down-regulate VEGFR2 expression in ECs exposed to VEGF through the histone methyltransferase KMT5B. Knockdown of endogenous KAP1 inhibits the TNF-α-induced phosphorylation of IKKα/β and IkBα, degradation of IkBα, and nuclear translocation of NF-kB/p65 in ECs [12]. We therefore speculate that KAP1 may also regulate the expression of LOX-1 in ECs after exposure to OxLDL. A recent study has shown that KAP1 is highly expressed in atherosclerotic plaque tissue [13]. These findings suggest that KAP1 may be a novel protein that participates in the pathological mechanisms of AS.

Given that OxLDL-induced ED is considered a pivotal event during the onset of AS and that the OxLDL/LOX-1 pathway has been implicated in the pathogenesis of ED, whether KAP1 plays a role in this process is unknown. Here, we tested the hypothesis that the silencing of KAP1 down-regulates LOX-1 expression during OxLDL-induced ED, thereby mediating the expression of downstream target genes. Our findings suggest that developing targeted strategies to inhibit KAP1 may prove useful in the prevention or treatment of AS.

EA.hy926 ECs were purchased from the American Type Culture Collection (Rockville, MD, U.S.A.) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, U.S.A.) with 10% (v/v) fetal bovine serum (FBS; Gibco, Carlsbad, U.S.A.) in a humidified incubator with 5% CO₂ and 95% air at 37°C. OxLDL was purchased from Yiyuan Biotechnologies (YB-002), Guangzhou, China. Cells were incubated with or without OxLDL (150 μg/ml) for 24 h as previously described [9,14].

The siRNA targeting KAP1 (si-KAP1) and negative control siRNA (si-NC) were obtained from GenePharma (Shanghai, China). Human KAP1 siRNA sequences were: 5′-ACAGGACAGAGAACAGAGCTT-3′ (sense) and 5′-GCUCUGUUCUCUGUC-CUGUTT-3′(antisense). The negative control sequences were: 5′-UUCUCCGAAC-GUGUCACGUTT-3′(sense)and 5′-ACGUGACACGUUCGGAGAATT-3′(antisense). The si-KAP1 or si-NC was transiently transfected into EA.hy926 ECs using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, U.S.A.) according to the manufacturer’s protocols. Taking six-well transfection for an example, briefly, cells were transfected when inoculated to 60–80% confluence. Then the medium was discarded, washed with phosphate buffer saline (PBS; NaCl, 137 mmol/l; KCl, 2.7 mmol/l; Na₂HPO₄·12H₂O, 8 mmol/l; NaH₂PO₄·2H₂O, 1.5 mmol/l) for three times and 750 μl Opti-MEM (Gibco, 31985) was added to each well. Next, lipofectamine 3000 reagent and siRNA were diluted with 125 μl Opti-MEM medium, respectively, for 5 min. After that, the two dilutions were mixed for 20 min. Finally, the 250 μl mixture was added into each well. Cells were incubated with transfection complexes for 6 h before the medium was changed. Twenty-four hours later, the cells were used for experiments.

The ROS detection was performed using the ROS Fluorescent Probe-Dihydroethidium (DHE) Kit (Vigorous, Biotechnology, Beijing). Specifically, 24 h after siRNA transfection, cells were treated with or without OxLDL (150 μg/ml) for another 24 h. Then, the ECs were washed three times with the preheated 37°C Krebs-Henseleit buffer (NaCl, 131 mmol/l; NaHCO₃, 20 mmol/l; CaCl₂, 2.5 mmol/l; HEPES, 20 mmol/l; KCl, 5.6 mmol/l; NaH₂PO₄, 1 mmol/l; MgCl₂, 1 mmol/l, glucose, 5 mmol/l, pH = 7.4). Next, a moderate amount of Krebs-Henseleit buffer containing DHE was added into wells. The wells were avoided light and incubated for 30 min. After incubation, cells were washed with preheated Krebs-Henseleit buffer for three times. Finally, images were taken under the Olympus IX73 inverted fluorescence microscope. It was excited with blue or green light and the ROS-positive cells throughout the nuclei were dyed red. The numbers of positive cells from randomly fields were counted under a microscope (Olympus IX73, magnification × 100) and analyzed using Image J 1.25 software (National Institutes of Health, Bethesda, U.S.A.).

Cell proliferation was detected by CCK-8 assay. Specifically, after transfecting with siRNA for 24 h, EA.hy926 ECs were seeded into 96-well dishes at a density of 1 × 10⁴ cells/well. After adherence, cells were treated with or without OxLDL (150 μg/ml) for another 24 h. Then the CCK-8 reagent (VICMED, VC5001) was mixed with cell culture medium in 1:10, and the mixture was added into each well and incubated for 2 h at 37°C. Finally, the microplate reader (Bio-Tek, Winooski, VT, U.S.A.) was employed to measure the optical density (OD) at 450 nm.

Transwell assay was performed to detect cell migration using transwell chambers (Corning Incorporated, Corning, NY, U.S.A.). Briefly, EA.hy926 ECs transfected with siRNA were digested with trypsin (Beyotime, C0201) and then seeded in the upper chamber (1 × 10⁵ cells/well) with serum-free medium containing OxLDL (150 μg/ml) or not, while 600 μl of medium containing 10% FBS was used as a chemoattractant in the lower chamber. After incubation at 37°C for 24 h, the non-migrated cells on the upper side of the membranes were wiped off with a cotton swab. Migrated cells on the lower side of the membranes were fixed with 4% paraformaldehyde for 15 min and stained with 1% Crystal Violet for 15 min. The numbers of stained cells from randomly fields were counted under a microscope (Olympus IX73, magnification × 200) and analyzed using ImageJ 1.25 software (National Institutes of Health, Bethesda, U.S.A.).

Cells were lysed in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing 1% protease inhibitor cocktail (VICMED, VPI012), and the cell lysates were then centrifuged at 14000 × g at 4°C for 15 min. Protein concentrations were quantified using the Bicinchoninic Acid protein assay kit (BCA; Beyotime, P0010). Total proteins (50 μg) were separated by SDS-PAGE and electrotransferred to polyvinylidenedifluoride membranes (PVDF; Millipore, Billerica, MA, U.S.A.). Then the membranes were blocked with TBST buffer (NaCl, 150 mmol/l; Tris, 10 mmol/l; Tween-20 0.05% (v/v) pH 7.6) containing 5% non-fat milk powder at room temperature for 2 h. The membranes were then probed overnight at 4°C with primary antibodies including phospho-KAP1(1:1000, Abcam, ab70369), MMP-9 (1:1000, Abcam, ab38898), KAP1(1:1000, Cell Signaling Technology, #4123), ICAM-1 (1:1000, Cell Signaling Technology, #4915), VCAM-1 (1:500, Affinity, DF6082) and PCNA (1:2000, Servicebio, GB11010), c-Myc (1:1000, Proteintech, 10828-1-AP), followed by incubation with corresponding HRP-conjugated secondary antibodies (1:5000, Proteintech, SA00001-2) at room temperature for 2 h. Proteins on the membranes were visualized by adding the enhanced chemiluminescence reagent (Millipore, U.S.A.). Band intensities were analyzed using ImageJ 1.25 software (National Institutes of Health, Bethesda, U.S.A.) and normalized to that of loading controls.

Total RNA was extracted using the RNAiso Plus(TaKaRa, Code No. 9109) and then reverse-transcribed into cDNA using a Reverse Transcription kit (Takara, Code No. RR036A). Quantitative real-time PCR was carried out by SYBR Green (Takara, Code No. RR820A) using the Roche LightCycler480 system (Roche, Nutley, NJ, U.S.A.). The reactions were performed according to the manufacturer’s protocols and the results were normalized to the Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) expression level. The whole melting curves contained single peaks, indicating a specific PCR amplification. The relative amount of each gene was calculated using the 2−ΔΔct as means of duplicate measurements. Supplementary Table S1 lists the primer sequences used in the present study.

All assays were performed at least in triplicate. Data were expressed as the mean ± SEM and analyzed using GraphPad Prism 7 statistical software. Statistical analysis was performed by Student’s t-test or one-way analysis of variance (ANOVA) test and P<0.05 was considered statistically significant.

Here, we first measured KAP1 expression in ED cell models induced by OxLDL. Western blot analysis revealed that the phosphorylation and protein levels were not significantly different between OxLDL-treated and non-treated ECs (Figure 1).

To further characterize the role of KAP1 in ECs treated with OxLDL, we utilized specific siRNA that could reduce the level of KAP1 in ECs. The efficacy of interference was ascertained by Western blot analysis and qRT-PCR (Figure 2A,B). Next, we measured the expression of pro-inflammatory vascular adhesion molecules, including ICAM-1 and VCAM-1, and found that KAP1 knockdown by siRNA (si-KAP1) significantly abolished the up-regulation of ICAM-1 and VCAM-1 expression in ECs induced by OxLDL (Figure 2C,D). Using DHE staining, we found that OxLDL treatment resulted in sharp intracellular ROS augmentation in ECs. However, KAP1 silencing ameliorated OxLDL-induced cellular ROS production (Figure 3).

We next characterized the role of KAP1 in the proliferation and migration of ECs. CCK-8 and transwell assays revealed that OxLDL-treated ECs demonstrated greater proliferation and migration. However, these alterations were reversed in KAP1 gene-silenced cells induced by OxLDL (Figure 4A,B). In addition, Western blots revealed that si-KAP1 repressed increases in proliferative and migratory markers, including c-Myc, PCNA, and MMP-9 in OxLDL-induced ECs (Figure 4C), further confirming the inhibitory effects of si-KAP1 on the proliferation and migration of ECs stimulated by OxLDL.

To determine whether si-KAP1 could affect LOX-1 expression under ED induced by OxLDL, Western blot analysis was performed using specific antibodies targeting LOX-1 and eNOS to demonstrate that the protection provided by KAP1 silencing against OxLDL insult was mediated through the down-regulation of LOX-1. The expression of LOX-1 increased and the expression of eNOS decreased in ECs treated with OxLDL. Nonetheless, KAP1 knockdown significantly reversed these alterations (Figure 5), which may partly explain the potential role of KAP1 in OxLDL-induced endothelium injury.

Overall, the results of our study allowed us to construct a brief schema illustrating the role of KAP1 deficiency in ECs. This schema demonstrates that knockdown of KAP1 might counteract OxLDL-induced ED, including the release of ROS, the increase in the expression of adhesion molecules, and abnormal endothelium behavior. Furthermore, this protection is likely correlated with the down-regulation of LOX-1 expression, which leads to the enhancement of antioxidation and the inhibition of aberrant endothelium behavior (Figure 6).

KAP1/TRIM28/TIF1β is a transcription regulatory factor in embryonic development located in the nucleus [12,15,16]. It is widely expressed in multiple tissues and organs, controls stem cell self-renewal, and is involved in chromatin remodeling and the DNA damage response [17]. Numerous studies have shown that KAP1 is highly abundant in malignant tumor cells or tissues, which is associated with the malignant behavior characteristic of the progression of tumors [18–21]. Some studies have also shown that KAP1 is closely related to the regulation of T cells in the immune system [22–24]. However, few studies have examined the role of KAP1 in atherosclerotic cardiovascular and cerebrovascular diseases. Recently, Liu et al. reported that KAP1 was highly expressed in human atherosclerotic tissues and cultured human aortic smooth muscle cells stimulated by platelet-derived growth factor subunit B homodimer [13]. Specifically, they found that knockdown of KAP1 suppressed the proliferation and migration of human aortic smooth muscle cells with atherosclerosis phenotypes, revealing that KAP1 plays a vital role in AS. In the present study, we demonstrated that the phosphorylation and protein level of KAP1 did not differ between OxLDL-treated and non-treated ECs (Figure 1). This pattern may stem from the complicated pathological process of atherosclerosis. After all, the induction of ED by OxLDL accounts for only one small component of the numerous factors contributing to AS. Although some irritants that mimic AS are capable of influencing the expression of KAP1, EC damage caused by OxLDL was not sufficient for inducing any changes in KAP1 phosphorylation and protein expression.

OxLDL-mediated injury to ECs is crucial for ED in the pathogenesis of AS, as well as atherosclerotic plaque rupture at advanced stages [25]. One clinical data analysis showed that serum OxLDL could independently predict pulse-wave velocity in subjects with normal or mildly reduced renal function [4]. Another study revealed that apple polyphenols could inhibit xanthine oxidase, thereby relieving vascular oxidative stress and improving endothelium function [5]. Thus, oxidative stress is closely correlated with cardiovascular and metabolic disease in patients with established renal disease. In addition, OxLDL-induced oxidative stress could activate the NF-κB signaling pathway and promote the expression of adhesion molecules in ECs [26].

In the present study, we showed that ECs treated with OxLDL for 24 h showed increased expression of ICAM-1 and VCAM-1, as well as ROS generation, patterns that are consistent with previous studies [9,26]. However, we found that knockdown of KAP1 suppressed the OxLDL-facilitated expression of ICAM-1 and VCAM-1 (both mRNA and protein levels) and ROS generation in ECs (Figures 2C,D and 3). Thus, KAP1 silencing can protect ECs against OxLDL-induced inflammation and oxidative stress. Moreover, OxLDL-elevated ROS in ECs could stimulate cell proliferation and migration, which is associated with unstable atheromatous plaque [27,28]. In addition, an increasing amount of evidence suggests that inflammation promoted endothelium proliferation and angiogenesis [29]. Cell proliferation and migration were both required for vascular formation, which was correlated with an increased blood supply in atheromatous plaque and resulted in increased sizes of the plaque and blockages in the blood vessels. Thus, abnormal EC behavior plays an important role in contributing to adverse atherosclerotic events. Here, we showed that OxLDL accelerated the proliferation and migration of ECs, while si-KAP1 abrogated this phenomenon (Figure 4A,B). Meanwhile, KAP1 silencing reversed the OxLDL-enhanced expression of two proliferative markers, c-Myc and PCNA, as well as the migratory marker MMP-9 (Figure 4C). Therefore, KAP1 deficiency can improve OxLDL-induced ED via modulation of the abnormal proliferation and migration of ECs, which has a protective function during AS.

Because it is a transcriptional regulatory factor, KAP1 is thought to be associated with the transcription of specific genes and the modification of protein post-translation. KAP1 is known to combine with STATs and interfere with IL-6-mediated signals in HeLa cells [30]. Moreover, KAP1 can inhibit IFN regulatory factor-mediated expression of TNF-α in macrophages [31]. Wang et al. showed that endogenous KAP1 depletion attenuated TNF-α-induced expression of VCAM-1 and ICAM-1 by inhibiting the expression of TNFR-1 and TNFR-2 in ECs [12]. We observed that KAP1 silencing alleviated OxLDL-induced increases in LOX-1 expression (Figure 5A). This finding suggested that the expression of LOX-1 may be regulated by KAP1. The binding of OxLDL to LOX-1 is known to rapidly increase ROS levels in human ECs [8]. Additionally, the activated oxidase stress can further increase LOX-1 levels [32]. The results are consistent with our data. OxLDL/LOX-1-dependent biological processes contribute to plaque instability and the ultimate clinical sequence of plaque rupture along with life-threatening tissue ischemia.

Over the past decades, multiple drugs, including natural antioxidants, anti-LOX-1 antibodies, and antisense oligodeoxynucleotide, have been used to inhibit AS by decreasing vascular LOX-1 expression and activity [25]. Previous studies have noted that the treatment of OxLDL impaired eNOS activity in ECs through LOX-1 activation [33]. In our study, eNOS expression was diminished in ECs after exposure to OxLDL for up to 24 h (Figure 5B). Strategies targeting the enhancement of eNOS activation resulted in increased protection against cell death [34]. Zhou et al. reported that long-term treatment (12–24 h) with OxLDL (100 µg/ml) up-regulated LOX-1 expression and down-regulated the level of eNOS, thereby mediating eNOS activity. Pre-treatment with JTX20 (a LOX-1 blocking antibody) effectively eliminated the ability of OxLDL to mediate the activation of signaling in bovine aortic ECs [35]. Another study has shown that ECs incubated with OxLDL (150 μg/ml) for 24 h markedly increased ROS generation, adhesion molecules and LOX-1 expression but decreased the level of eNOS [14], which is consistent with our findings (Figure 6). Nevertheless, pre-incubation with Klotho (a type of anti-aging protein) effectively prevented OxLDL from affecting any of these alterations. Additional research has revealed that Klotho could alleviate OxLDL-mediated oxidative stress in human umbilical vein ECs via activation of the PI3K/Akt/eNOS pathway and depression of LOX-1 expression [36]. Therefore, LOX-1 represents a promising therapeutic target for the treatment of human atherosclerotic diseases [37].

Although OxLDL-induced ED is considered an important event during AS progression and the OxLDL/LOX-1/eNOS pathway has been implicated in the pathogenesis of ED, there is still a lack of information on whether KAP1 plays a role in this process. In the present study, we tested the hypothesis that si-KAP1 could influence LOX-1 expression during OxLDL-induced ED. KAP1 knockdown significantly down-regulated the expression of LOX-1 but promoted eNOS expression in ECs treated with OxLDL (Figure 5).

Here, we explored the role of KAP1 in OxLDL-induced ED and found that KAP1 silencing protected the endothelium from oxidation damage possibly by inhibiting LOX-1 expression. However, the role of the LOX-1 pathway in mediating the effect of si-KAP1 on ECs remains unclear. Furthermore, other types of cell lines, such as SMCs and macrophages, which are closely associated with atherosclerosis, need to be studied. In addition, an in vivo experiment would aid our understanding of the role of KAP1 in the development of AS. Finally, the relationship between KAP1 and the clinical features of AS need to be explored.

In sum, knockdown of KAP1 may protect ECs from OxLDL-mediated injury by depressing the expression of LOX-1 and up-regulating the expression of eNOS, thus participating in the onset and development of AS. Therefore, our study provides new insight into how targeted strategies for depleting KAP1 could be used to control the proatherogenic effects mediated by OxLDL/LOX-1. Specifically, the development of a KAP1 inhibitor or LOX-1 blocker could prove particularly useful for the prevention or treatment of AS.

The authors declare that there are no competing interests associated with the manuscript.

This study was supported by grants from the Specialized Research Fund for Senior Personnel Program of Xuzhou Medical University [grant number D2018015]; the Xuzhou Science and Technology Planning Project [grant number KC18052]; and Jiangsu Provincial Scientific Research Innovation Planning for Graduate Students [grant number KYCX19_2211].

M.P. and W.W.: Study design. Y.T.Q.: Measurement of ROS production, cellular viability and migration analysis. L.C.: Protein expression study. Y.T.Q.: Manuscript drafting. L.C.: Data and statistical analysis. F.H.D., Z.W., H.L.Y. and Q.S.H.: Manuscript revision. All authors listed have approved this manuscript.

AS

atherosclerosis

CCK-8

Cell counting kit-8

EC

endothelial cell

ED

endothelial dysfunction

ICAM-1

intercellular adhesion molecule-1

KAP1

KRAB domain-associated protein 1

KRAB-ZFP

Kruppel-associated box zinc finger protein

LOX-1

lectin-like oxidized low-density lipoprotein receptor-1

MMP-9

matrix metalloproteinases-9

OxLDL

oxidized low-density lipoprotein

PCNA

proliferating cell nuclear antigen

ROS

reactive oxygen species

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TIF1β

transcriptional intermediary factor 1 beta

TRIM28

triple motif protein 28

VCAM-1

vascular cell adhesion molecule-1