Radiation‐induced C‐reactive protein triggers apoptosis of vascular smooth muscle cells through ROS interfering with the STAT3/Ref‐1 complex

Abstract Damage to normal tissue can occur over a long period after cancer radiotherapy. Free radical by radiation can initiate or accelerate chronic inflammation, which can lead to atherosclerosis. However, the underlying mechanisms remain unclear. Vascular smooth muscle cells (VSMCs) proliferate in response to JAK/STAT3 signalling. C‐reactive protein (CRP) can induce VSMCs apoptosis via triggering NADPH oxidase (NOX). Apoptotic VSMCs promote instability and inflammation of atherosclerotic lesions. Herein, we identified a VSMCs that switched from proliferation to apoptosis through was enhanced by radiation‐induced CRP. NOX inhibition using lentiviral sh‐p22phox prevented apoptosis upon radiation‐induced CRP. CRP overexpression reduced the amount of STAT3/Ref‐1 complex, decreased JAK/STAT phosphorylation and formed a new complex of Ref‐1/CRP in VSMC. Apoptosis of VSMCs was further increased by CRP co‐overexpressed with Ref‐1. Functional inhibition of NOX or p53 also prevented apoptotic activity of the CRP‐Ref‐1 complex. Immunofluorescence showed co‐localization of CRP, Ref‐1 and p53 with α‐actin‐positive VSMC in human atherosclerotic plaques. In conclusion, radiation‐induced CRP increased the VSMCs apoptosis through Ref‐1, which dissociated the STAT3/Ref‐1 complex, interfered with JAK/STAT3 activity, and interacted with CRP‐Ref‐1, thus resulting in transcription‐independent cell death via p53. Targeting CRP as a vascular side effect of radiotherapy could be exploited to improve curability.


| INTRODUC TI ON
Radiotherapy is prescribed for many malignancies, including over 50% of solid cancer patients, resulting in high survival rates for patients with early head and neck cancer or breast cancer. [1][2][3] However, radiotherapy can cause many years later chronic inflammatory diseases affecting the vascular system, including stroke, myocardial infarction, acute artery rupture and unstable atherosclerotic plaques. 4,5 In chronic inflammation, vascular smooth muscle cells (VSMCs) undergo a phenotypic switch that involves cell proliferation, migration, death and senescence in blood vessel walls, which can proceed over several decades. 6 Furthermore, VSMCs-specific loss can promote a variety of inflammatory responses in blood vessels, including increased macrophage recruitment. A phenotypic switch of VSMCs from proliferation to apoptosis or cell senescence can result in advanced atherosclerosis or vascular damage, notably through plaque instability, leading to a critical condition called acute coronary syndrome. 6 The fibrous cap weakened by VSMCs loss is also a major factor in the ruptured-or vulnerable plaques formation. [5][6][7][8] Providing a mechanistic understanding of the molecular mediators able to modify this phenotype is important to prevent side effects and improve radiotherapy.
Because the marker of inflammation C-reactive protein (CRP) is sensitive to radiation, serum CRP levels are measured in biodosimetry following radiation accidents. 9 Blood CRP values of 30 rhesus monkeys were increased in a dose-and time-dependent manner upon exposure to 1-8.5 Gy of 60 Co γ-rays. 10 The level of CRP reflects the time course and inflammatory severity of radiotherapy and can predict the outcomes of cancer patients. 11 A high level of CRP is also a strong predictor of atherosclerosis and is closely associated with plaque instability histologically and clinically. [12][13][14] CRP is expressed in apoptotic artery VSMCs, 15 where it contributes to plaque rupture through the upregulation of NADPH oxidase (NOX). 16 In a previous study, we found that CRP can induce apoptosis of VSMC via NOX4 activation. 17 However, the pathophysiological mechanisms underlying the involvement of elevated CRP in the development of vulnerable plaques and the loss of VSMC remain unclear.
A ubiquitous human AP-endonuclease/Redox factor-1 (APE/Ref-1) plays multifunctional roles including repairing damaged DNA via the base excision repair pathway and regulating as a co-factor different transcriptional factors in controlling different cellular processes such as apoptosis, proliferation and differentiation. 18,19 Ref-1 was shown to inhibit excessive ROS production related to NOX and NF-κB activation. 18 Additionally, Ref-1 was found to be implicated in cardiovascular diseases. [19][20][21] Ref-1 also contributes to vessel wall changes from the Go/G1 to S phase in VSMC. 22 15,17,24,25 but their interactions remain unclear.
Vascular smooth muscle cells loss by apoptosis is a critical factor in driving vulnerable plaque formation by affecting vessel remodelling, inflammation and coagulation. 7 However, in numerous studies, VSMC proliferation has also been implicated in atherosclerosis and restenosis. 6,26 Blocking VSMC proliferation has been proposed as a therapeutic strategy by many studies. 26,27 Consequently, both the proliferation and apoptosis of VSMCs are associated with atherosclerosis exacerbation. So far, the molecular mechanisms responsible for vessel wall damage in the chronic inflammation that follows radiation therapy have not been elucidated. were cultured in DMEM medium with 1500 mg/L sodium bicarbonate (Gibco BRL) in a 5% CO 2 /37°C incubator.

| Analysis of apoptotic genes mRNA expression
Specific sequences for PCR were amplified using the primers and number of cycles (94°C for 30 s, 58°C for 40 s and 72°C for 1 min) listed in Table S1.

| Ionized radiation (IR)
The monolayers on flasks were irradiated with 6MV photons produced by a medical linear accelerator (Varian Clinac 21EX; Varian).
Luminescence dosimeters (nanoDOT; Landauer) were used to measure the dose delivered by the Varian linear accelerator. Calibration of the dosimeters was performed using a MicroStar InLight reader (Landauer), and the error was <1% compared with the planned values for the cell lines (hVSMCs and A10 cells) at 2, 4 and 8 Gy. 30

| Staining of Annexin V and propidium iodide (PI)
IR-exposed or IR-overexpressed CRP in A10 cells (1 × 10 5 cells per four-well cultured plate) were measured using the Annexin V-FITC apoptosis detection kit (CELLQUEST software; BD Pharmingen) after 16 or 24 h. Positive staining was screened using a confocal microscopy system (LSM710 Carl Zeiss GmbH).

| Preparation of cytoplasmic and nuclear extracts
Nuclear extracts were prepared as described previously. 31 Briefly, Nuclei were collected by centrifugation at 2,860 g at 4°C for 10 min. The supernatant was considered the cytoplasmic fraction.
Cytoplasmic extracts were subsequently centrifuged at 11,460 g for 20 min at 4°C.

| Isolation of mitochondria
Mitochondrial fractions were isolated from 2 × 10 7 VSMCs using the Mitochondria Isolation Kit (Thermo Scientific Pierce Biotechnology) according to the manufacturer's instructions.

| Immunoblotting (IB) assay
Western blot was performed using primary antibodies against the proteins of interest as previously described. 17 The antibodies are listed in Table S2. The protein amount was quantified by scanning photo-densitometry and its quantitation software (MULTI-IMAGE & Bio-Rad Laboratories Inc.).
The immunoprecipitants were subjected to Western blotting using the indicated antibodies. 17

| Immunofluorescence (IF)
Fluorescence-tagged secondary antibodies were used to reveal the primary antibody (Table S2)

| Statistical analysis
The SPSS package was used to perform the statistical analyses. The results are shown as mean ± standard deviation (SD) of three independent experiments. Means for different groups were compared using unpaired, two-tailed Student's t-tests (***p < 0.001, **p < 0.01, *p < 0.05).

| Ionizing radiation enhances ROS production, CRP and Ref-1 expression in VSMCs
In radiotherapy, cellular damage is caused by ionized radiation, generating a variety of ROS. 2,32 In general, ROS generation by radiation disappears momentarily. However, intracellular ROS can be continuously generated for 48 h and longer by activation of NOXs through secondary cell signalling due to radical stress in blood vessel cells. 33 IR-exposed VSMCs produced intracellular ROS dose-dependently even after 48 h. Mean fluorescence intensity (MFI) of intracellular ROS in 0 G, 4 Gy and 8 Gy IR-exposed VSMCs was 12.18 ± 2.5, 20.20 ± 1.2 and 25.88 ± 1.7 respectively ( Figure 1A,B). Low-dose cellular exposure to IR (0.1-10 Gy) can induce mitochondrial dysfunction via an accumulation of mitochondrial ROS. 32 The 8 Gyexposed VSMCs produced significantly more mitochondrial ROS at 48 h. Mitochondrial ROS MFI of 0 Gy-and 8 Gy-exposed VSMCs were 50.87 ± 13.89 and 128.31 ± 34.11 respectively, which were two times higher than the control ( Figure 1C,D). CRP is also a biomarker of biodosimetry, 34 as its accumulation in patients undergoing radiotherapy can reflect various chronic inflammatory diseases. 35 In IR-exposed VSMC, radiation dose-dependently increased the CRP mRNA level after 24 h ( Figure 1E). The expression levels of Ref-1, which is known to regulate redox homeostasis, 20 were further increased by radiation ( Figure 1E-G). IR also dose-dependently increased the mRNA levels of NOX4, GADD153 and Bax ( Figure 1E).
The expression of p22 phox , which is a major subunit of NOXs, 36

| Ionizing radiation enhances apoptosis of VSMCs through ROS production by NOX
Measuring ROS production from NOXs is particularly relevant to understanding the roles of ROS in the physiology and pathophysiology of VSMCs. 37 (Figure 2A-C). To determine whether ROS production through IR-induced NOX enhanced VSMC apoptosis, apoptosis was measured under the same conditions after pretreatment with DPI or NAC. The apoptosis of VSMCs was found to be reduced by about 50% upon NOX-specific inhibition of DPI (4 Gy-exposed cells vs. DPI pretreated 4 Gy-exposed cells: 48.44 ± 3.04 vs. 25.59 ± 7.077; 8 Gyexposed cells vs. DPI pretreated 8 Gy-exposed cells: 61.82 ± 9.53 vs. 29.99 ± 9.03). Apoptosis was further attenuated following pretreatment with NAC, a ROS scavenger, in human VSMCs (4 Gyexposed cells vs. NAC pretreated 4 Gy-exposed cells: decreasing 32%; 8 Gy-exposed cells vs. NAC pretreated 8 Gy-exposed cells: decreasing 41%) (Figure 2A-C). IR exposure also increased caspase-3 activity more than three times (CON vs. 8 Gy: 120% ± 20% vs. 320% ± 40%). However, the IR-induced caspase-3 activity decreased by 47.1% following 1 μM DPI pretreatment and by 77% following 500 μM NAC pretreatment compared with the control group ( Figure 2D). These results suggest that apoptosis can lead to an inhibition of caspase-3 activity through the depletion of ROS. In VSMCs from atherosclerosis patient samples, p22 phox is an essential component of isotypes of NOXs. 38 It has been shown that p22 phox deficiency can inhibit activation of NOXs. 17,28,36,39 To confirm this finding, a stable p22 phox -knockdown VSMC (p22 phox KD) was constructed ( Figure S1). Confocal images showed that the IR-induced DNA damage in VSMCs was also markedly reduced in p22 phox KD ( Figure 2E). These results overall suggest that IR induces apoptosis by increasing ROS production of NOX in VSMCs.

| Ionized radiation increases VSMCs apoptosis through CRP-mediated ROS production
Our previous study revealed that CRP, as a ligand of the Fcγ receptor IIA (FcγRIIA), can induce VSMCs apoptosis via ROS production by NOX4. 17 To assess the effect of CRP accumulation, human CRP was overexpressed in A10 cells (tCRP) ( Figure S2).
At 24-hours' post-transfection, the cells were analysed to detect apoptosis ( Figure S3). Confocal microscopy showed that tCRP critically induced apoptosis and nucleic DNA damage in A10 cells ( Figure 3A). The tCRP-A10 cells showed increased production of intracellular ROS (705.4% ± 66.2% compared to 100% ± 15.1% for control tMOCK-A10 cells) ( Figure 3B). The mean value of specific mitochondrial ROS was also shifted from 9.14 (tMock) to 16.4 (tCRP) in A10 cells ( Figure 3C). To confirm that CRP protein leads to ROS production, mRNA levels of redox-related genes were de-

Ref-1 complex and alters its expression levels in cytoplasmic and nuclear fractions
Ref-1 can bind to STAT3 and act as its transcriptional co-factor in the nucleus. 20

| The CRP/Ref-1 complex changes cause VSMCs death via cytoplasmic p53
As shown in Figure 6A-C, tCRP in VSMCs increased the expression levels of apoptotic genes, including p53, bax, bak and cleaved caspase-3, whereas tRef-1 alone increased Bcl2. This suggests that CRP might change the phenotype of VSMCs. Ref-1 protein is associated with p53. 18,40 To examine this possibility, IP was performed using myc-tagged CRP-overexpressed VSMCs. The myc-tagged tCRP induced-p53 formed a complex with Ref-1/CRP proteins in VSMCs ( Figure 6B). As shown in Figure 4B,C, co-overexpressed CRP and Ref-1 in VSMCs were markedly increased in the cytoplasm.
Mono-ubiquitination of p53 causes it to transmigrate from the nucleus to the cytoplasmic compartment. 41 As shown in Figure 6B, mono-ubiquitinated p53 was mainly detected in the lane with tCRP-VSMC. The tRef-1 alone could not induce mono-ubiquitination of p53. tCRP induced p53 expression and co-localized with p53 in the mitochondria or cytoplasm ( Figure S6). When p53 migration was blocked using pifithrin μ (PFT μ), an inhibitor of cytoplasmic accumulation of p53, 42 the expression levels of Bax and Bak were attenuated compared with those in tCRP-VSMCs ( Figure 6C). In addition, confocal images showed that CRP-induced cell death was blocked by inhibiting cytoplasmic p53 using PFT μ or p53 siRNA ( Figure 6D,E).
These results suggest that the complex formation of CRP and Ref-1 in VSMCs might increase apoptosis via the accumulation and association with cytoplasmic p53.

| CRP, Ref-1 and p53 proteins are co-localized in VSMCs of human coronary plaques
Human atherosclerotic plaques contain abundant VSMCs and macrophages. 43 The image of immunofluorescence clearly showed that

| DISCUSS ION
Radiation-induced CRP initiates and maintains chronic inflammation that causes late radiation complications. The serum CRP level is usually measured from undetectable to 1.0 mg/L in healthy humans. CRP can be de novo synthesized in VSMCs or macrophages within atherosclerotic lesions in inflammatory responses. 44 Blaschke et al. 15 reported that CRP can induce apoptosis of human coronary VSMCs by mediating caspase activity. In addition, we have previously reported that CRP can trigger NOX4 activation and consequently induce apoptosis of human aortic VSMCs. 17 Radiotherapy-induced CRP can increase the risk of skin toxicity and cardiac morbidity, and ischaemic heart diseases. 45 In this context, the present study confirmed that IR-exposed VSMCs produced ROS dose-dependently even 48 h later. In artificial conditions, overexpressed CRP in VSMCs dramatically increased their intracellular ROS production.
To examine how VSMCs survival could be modulated by de novo synthesis of CRP, hrCRP was used to treat VSMCs. This Localization of mitochondria and overexpressed CRP protein in A10 cells was analysed through IF using MitoTracker, rabbit-anti-CRP antibody and FITCconjugated secondary anti-rabbit antibody. Nuclei were stained with DAPI. Magnification, 630×. CRP expression pattern and localization of mitochondria are shown using confocal microscopic images (panels a and e: the nucleus is blue, panels b and f: CRP is green at 488 nm, panel c and g: mitochondria is red at 578 nm) NAC in IR-exposed VSMCs reduced the expression of apoptosis genes. CRP in the region of directional coronary atherosclerosis (DCA) can induce ROS production from p22 phox -based NOX. 17,48 Furthermore, NOX1, NOX2 and NOX4 proteins strictly require the p22 phox subunit for their enzyme activity. 28,36 We generated p22 phox knockdown of VSMCs (p22 phox -KD) by lentivirus to inhibit NOX activity, which was able to inhibit the induction of p22 phox expression by CRP ( Figure S7). We also suppressed NOX activity using DPI. Both approaches consistently demonstrated profound inhibition of radiation-induced apoptosis by attenuating caspase-3 activity and DNA damage. These results confirmed that radiationinduced CRP could increase ROS production by NOX, thus leading to apoptosis of VSMCs.
In the early phase of atherosclerosis, VSMCs affects some as-   56 Here, we showed that overexpressed CRP of VSMCs dramatically enhanced p53 expression and the proportion of monoubiquitinated p53 under the same conditions. Mono-ubiquitination of p53 triggers its export from the nucleus. 41 Nuclear exported p53 can accumulate in the cytoplasm before transcription-independent p53 apoptosis via mitochondrial dysfunction, cytochrome c release and procaspase-3 activation. 41,42 When mono-ubiquitinated p53 was associated with the CRP/Ref-1 complex in VSMCs, we found that the expression levels of apoptotic genes, including Bax, Bak, cleaved caspase-3 and cytosolic cytochrome C were increased.
Exporting p53 from the nucleus might play a pro-apoptotic role.
However, pretreatment with PFT μ or p53 siRNA, an inhibitor of mitochondrial p53 accumulation and function, 42

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

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this published article and its supplementary information files.