Protein deglycase DJ‐1 deficiency induces phenotypic switching in vascular smooth muscle cells and exacerbates atherosclerotic plaque instability

Abstract Protein deglycase DJ‐1 (DJ‐1) is a multifunctional protein involved in various biological processes. However, it is unclear whether DJ‐1 influences atherosclerosis development and plaque stability. Accordingly, we evaluated the influence of DJ‐1 deletion on the progression of atherosclerosis and elucidate the underlying mechanisms. We examine the expression of DJ‐1 in atherosclerotic plaques of human and mouse models which showed that DJ‐1 expression was significantly decreased in human plaques compared with that in healthy vessels. Consistent with this, the DJ‐1 levels were persistently reduced in atherosclerotic lesions of ApoE−/− mice with the increasing time fed by western diet. Furthermore, exposure of vascular smooth muscle cells (VSMCs) to oxidized low‐density lipoprotein down‐regulated DJ‐1 in vitro. The canonical markers of plaque stability and VSMC phenotypes were evaluated in vivo and in vitro. DJ‐1 deficiency in Apoe−/− mice promoted the progression of atherosclerosis and exaggerated plaque instability. Moreover, isolated VSMCs from Apoe−/−DJ‐1−/− mice showed lower expression of contractile markers (α‐smooth muscle actin and calponin) and higher expression of synthetic indicators (osteopontin, vimentin and tropoelastin) and Kruppel‐like factor 4 (KLF4) by comparison with Apoe−/−DJ‐1+/+ mice. Furthermore, genetic inhibition of KLF4 counteracted the adverse effects of DJ‐1 deletion. Therefore, our results showed that DJ‐1 deletion caused phenotype switching of VSMCs and exacerbated atherosclerotic plaque instability in a KLF4‐dependent manner.


| INTRODUC TI ON
Atherosclerosis, the primary cause of cardiovascular disease and the top cause of death globally, is a complex disease initiated by the accumulation of lipids in the subendothelial layer of the arterial wall. [1][2][3][4] The most important clinical consequence of atherosclerosis is acute rupture or erosion of unstable plaques in which vascular smooth muscle cells (VSMCs) constitute the primary cell type; this finding has been verified by lineage-tracing experiments and single-cell sequencing. [5][6][7][8][9][10][11] During this pathological process, VSMCs change from a contractile phenotype to a de-differentiated phenotype (also called a synthetic phenotype), marked by reductions in various unique contractile proteins (eg α-smooth muscle actin [SMA], calponin1, smooth muscle 22α and smooth muscle myosin heavy chain) and higher rates of migration and proliferation. 8,[12][13][14][15][16][17] Many studies have evaluated the mechanisms and factors involved in VSMC phenotype switching.
Protein deglycase DJ-1 (DJ-1), a 189-amino acid protein (also known as PARK7), was first identified as an original oncogene accountable for the autosomal recessive early-onset form of Parkinson's disease. [18][19][20][21] As a multifunctional protein, DJ-1 is ubiquitously distributed and play essential roles in various biological process. 22,23 Although DJ-1 is positively associated with tumours, several studies have found that DJ-1 protects cells from different harmful stimuli. For example, mutations in DJ-1 result in neurodegeneration, leading to an early-onset familial form of Parkinson's disease. 20,24,25 Moreover, overexpression of DJ-1 defends against oxidative stress-induced damage, whereas models of the absence of DJ-1 are more susceptible to cerebral ischaemia, neuronal cell death, hypertension and obesity owing to increased oxidative stress. 24,[26][27][28][29] Using proteomics analysis, Won et al showed that DJ-1/PARK7 responds rapidly to oxidative stress in VSMCs. 30,31 Subsequently, they demonstrated that DJ-1 inhibits neointimal hyperplasia and maintains vasorelaxation by participating in the synthesis of endothelial nitric oxide synthase. 32 Altogether, these findings denoted that DJ-1 has vital functions in the vascular system. However, the association between DJ-1 and atherosclerosis and the direct mechanisms through which DJ-1 affects VSMC phenotype switching remains unclear.
Accordingly, in this study, we evaluated the impact of DJ-1 deletion on atherosclerotic plaque stability and clarified the potential mechanisms.

| Materials
The following primary antibodies were used in this study: anti-

| Animal experiments
All animal experiments were completed regarding the rules approved by the Institutional Committee for the Use and Care of Laboratory Animals of Qilu Hospital, Shandong University. ApoE -/mice were purchased from Beijing Viewsolid Biotech Co; Ltd.

| Tissue harvesting and preparation
Mice were anaesthetized at different time-points by intraperitoneal injection of pentobarbital natrium (40 mg/kg bodyweight).
We peeled adherent adipose tissue off the whole aortas and aortic roots. The aortas and aortic roots were then immediately fixed in 4% paraformaldehyde for subsequent experiments. Next, aortic root samples were embedded using OCT (Fisher, Tustin, CA, USA) or paraffin and serially sectioned at 8 μm thickness.

| Human tissue harvesting
Human coronary arteries were obtained from Qilu Hospital of Shandong University based on the protocols for human studies approved by the institutional ethical committee.

| Immunohistochemistry
Tissue samples were incubated with primary antibodies at a dilution of 1:200. After that, they were incubated with a goat anti-mouse/rabbit peroxidase-labelled antibody (ZSGB-BIO) as a secondary antibody for 30 minutes. Histopathological slide analysis was performed with ImagePro Plus software.

| Immunofluorescence staining of sections
The immunofluorescence staining was examined using 7-mm frozen sections of the aortic roots. The sections were blocked with 5% normal donkey serum (Dako) at room temperature for 1 hour, followed by the incubation of primary antibodies (1:200) at 4°C overnight. After 1 hour incubation with the second antibody at room temperature, the sections were then washed three times with phosphate-buffered saline (PBS), and the nucleus was co-stained with 4′,6-diamidino-2-phenylindole (DAPI).

| Cell culture and Small interfering RNA (siRNA) transfection
Primary mouse VSMCs were isolated from aortae of 8-week-old mice (from the aortic roots to bifurcation of the renal arteries) as described. 33 VSMCs were cultured in complete Dulbecco's modified Eagle's medium at 37°C in an atmosphere containing 5% CO 2 .
The transient transfection of siRNA into VSMCs was achieved using Lipofectamine RNAi MAX transfection reagent (Thermo Fisher Scientific) according to the manufacturer's instructions.
Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) Total RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and reverse-transcribed into cDNA using the Prime Script RT reagent Kit (Takara, Dalian, China). Individual qRT-PCR was carried out using specific primers, as described in the Supplement files.

| Statistical analysis
Data are expressed as means and standard errors of the means.
Statistical analysis was carried out through one-way analysis followed by Tukey's tests with SPSS statistical software package (version 22.0; SPSS, Chicago, IL, USA). Results with P values of less than 0.05 were considered significant.

| DJ-1 expression was reduced in human atherosclerotic carotid arteries
To prove whether DJ-1 expression was involved in atherosclerosis, we detected the expression of DJ-1 in healthy human vessels and carotid atherosclerotic plaques using immunochemistry and immunofluorescence staining. As shown in Figure 1A, the expression of DJ-1 was apparently lower in atherosclerotic plaques, which suggested a crucial role of DJ-1 in the progress of atherosclerotic plaques.

| Time-dependent decrease of DJ-1 in ApoE −/− mice fed by western diet
Next, we assessed DJ-1 expression in ApoE −/− mice with a western diet for different times. As illustrated in Figure 1B

| DJ-1 was down-regulated by ox-LDL in VSMCs in vitro
VSMCs are one of the vital constitutes in the development of atherosclerosis. 6,7,34 To assess the influence of atherosclerosis on DJ-1 expression in VSMCs, VSMCs were cultured with the atherosclerotic stimulus ox-LDL. Immunofluorescence imaging showed that DJ-1 expression was continuously down-regulated over time following stimulation with ox-LDL ( Figure 2A). Consistent with this, as shown in Figure 2B,C, DJ-1 protein levels were decreased to 64% after 12 hours of ox-LDL treatment in VSMCs and were further reduced to 42% after 24 hours of ox-LDL treatment. Similar findings were observed using qRT-PCR ( Figure 2D). These findings further demonstrated that DJ-1 expression in VSMCs was closely related to the development of atherosclerosis.

| DJ-1 deletion exacerbated atherosclerotic plaque instability
To further elucidate the role of DJ-1 in plaque stability, we use the Sirius red, Masson, CD68 and α-SMA staining. 35,36 As shown in

| DJ-1 deficiency induced VSMC phenotypic switching
Aberrant proliferation and phenotype switching of VSMCs contribute to the progression of atherosclerosis. 6,8,16,37 As demonstrated by immunohistochemical analysis (Figure 4A

| DJ-1 deficiency induced VSMC phenotype switching via the ERK/KLF4 pathway
KLF4, a known transcription factor of KLF family, is a critical negative regulator of VSMC contractile proteins. 38,39 Therefore, we reasoned that DJ-1 deletion may increase KLF4 expression to promote VSMC phenotypic switching and plaque instability.
To validate the hypothesis, we measured KLF4 expression in plaques from aortic roots and VSMCs. As shown in Figure 6A Figure   S2B). To explore the mechanism of how DJ-1 influences KLF4 expression, we focused on the common upstream kinases in the KLF4 signalling pathway, such as ERK1/2, AKT. [40][41][42][43] To confirm the involvement of ERK1/2 and AKT in DJ-1 effects on KLF4 expression in VSMCs, we firstly analysed the expression of ERK1/2, AKT and their phosphorylated form among which significant increased EKR1/2 were found in DJ-1 deficiency VSMCs ( Figure 6D). We further use ERK1/2 inhibitor to verify the hypothesis and found ERK1/2 inhibitor could effectively ameliorate the DJ-1 deficiency caused up-regulated KLF4 and inhibit the VSMCs phenotype transition towards synthetic type ( Figure 6E). Therefore, we conclude that DJ-1 prevented the synthetic alteration of VSMCs via KLF4 in an ERK1/2-dependent way. (Figure 7).

| D ISCUSS I ON
In this study, we found that DJ-1 deficiency accelerated the develop-

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.