Ring finger protein 10 is atherosclerosis protective and modulates macrophage polarization

Abstract

Macrophages can develop into pro-inflammatory M1-like macrophages and anti-inflammatory M2-like macrophages when stimulated by distinct internal environment. Dynamic changes of the two kinds of macrophages play key roles in atherosclerosis progression. The study aims to explore the role of ring finger protein 10 (RNF10) in regulating macrophage polarization during atherosclerosis. Mice with macrophage-specific depletion of RNF10 (RNF10^Mac-KO/ApoE^−/−) and control mice (RNF10^fl/fl/ApoE^−/−) mice were fed with high-fat diet to generate atherosclerotic lesion, from which peritoneal macrophages were isolated and transfected with RNF10-overexpressing vector. Murine macrophages, RAW264.7, were transfected with RNF10-overexpressing vector or RNF10 siRNA and stimulated with oxidized low-density lipoprotein (ox-LDL) to induce foam cell formation. The RNF10^Mac-KO/ApoE^−/− mice showed greater atherosclerotic lesions, more resident macrophages, higher expression of iNOS (M1-like macrophage marker), and lower expression of Arginase-1 (M2-like macrophage marker) than the RNF10^fl/fl/ApoE^−/− mice. RNF10 overexpression could reduce expressions of IL-1β, IL-6, and iNOS (M1 marker genes), increase expressions of IL-10 and Arg-1 (M2 marker genes) in the peritoneal macrophages isolated from RNF10^Mac-KO/ApoE^−/− mice. RNF10 overexpression reduced lipid accumulation in ox-LDL-induced foam cells, whereas RNF10 silencing yielded opposite results. Our data suggest that RNF10 is associated with M1-like macrophage suppression and M2-like increase, indicating RNF10 in macrophages has an anti-atherosclerotic role.

Introduction

Atherosclerosis is a chronic inflammatory disease progressively driven by lipid retention in the large-sized arteries leading to narrowing and thrombosis [1]. The development and rupture of atherosclerotic plaques remain the main pathology of cardiovascular diseases, and targeting the microenvironment of vulnerable plaques becomes an emerging diagnosis and therapy strategy for atherosclerosis [2]. Monocyte/macrophage functions responding to a variety of stimuli in the plaque microenvironment play an important role in the dynamic evolution of the plaque [3]. Macrophages not only engorge oxidized low-density lipoprotein (ox-LDL) to form foam cells accumulated within sub-endothelial intima but also secrete abundant proinflammatory factors to induce excessive inflammatory response, both of which can differentially affect the stability of the plaque [4, 5]. Macrophages can develop into classically activated M1 macrophages and alternatively activated M2 macrophages when stimulated by distinct internal environments [6]. M1-activated macrophages initiate and sustain inflammation and M2-activated macrophages resolve inflammation and remove cellular debris after apoptosis to repair tissue [7]. Accordingly, there has been a growing body of scientific evidence suggesting reducing atherosclerotic plaque formation or enhancing plaque stability by changing the number of macrophages [8].

Ring finger protein 10 (RNF10), first discovered in 2000, is a member of the RING (really interesting new gene) finger protein family which function as ubiquitin-protein E3 ligases [9, 10]. RNF10 has been extensively studied for its function in several biological processes, such as hyperproliferation, apoptosis, cell differentiation, and neuronal development [11,12,13,14]. RNF10 has also been reported to confer cardioprotection by its anti-inflammatory and anti-apoptotic effects in cardiomyocytes [15, 16]. Reduced RNF10 expression in macrophages may be involved in age-related inflammatory diseases [17]. Recently, a coronary plaque sampling approach has been developed and it revealed molecular insights into human coronary atherosclerosis, including RNF10 differentially expressed between stable coronary artery disease and acute coronary syndrome [18]. Given the positive linkage between RNF10 and cardiac inflammation, we proposed a hypothesis of macrophage RNF10 protection against atherosclerosis. To test this hypothesis, we generated macrophage-specific RNF10 knockout mice fed with high-fat diet and determined inflammatory state and the status of M1- and M2-like macrophages.

Materials and methods

Animals and atherosclerosis induction

C57BL/6J mice homozygous for the floxed RNF10 allele (RNF10^fl/fl) and C57BL/6J mice containing the Lyz2-Cre transgene were both obtained from Hunan SJA Laboratory Animal Co., Ltd. (Hunan, China). To generate macrophage-specific knockout mice (designated RNF10^Mac-KO), RNF10^fl/fl mice were bred with Lyz2-Cre mice. To generate mice with a double knockout of ApoE and RNF10 (RNF10^Mac-KO/ApoE^−/−), and littermate control (RNF10^fl/fl/ApoE^−/−), RNF10^fl/fl and RNF10^Mac-KO mice were bred with C57BL/6J mice with ApoE knockout (ApoE^−/−), respectively. The ApoE^−/− mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All mice were specific pathogen-free and housed in individually ventilated cages (Suzhou Houhuang Animal Laboratory Equipment Technology Co., Ltd., Jiangsu, China) with free access to water and food. They were maintained under a light-controlled room (12 h light/dark cycle) with a temperature of 22 ± 1°C and a relative humidity of 55 ± 10%. All mice The RNF10^Mac-KO/ApoE^−/− (5 males and 5 females) and RNF10^fl/fl/ApoE^−/− mice (5 males and 5 females) at 6–8 weeks of age were fed a high-fat diet (HFD) for 12 weeks to induce atherosclerotic lesion. All mice were euthanatized sodium pentobarbital anesthesia. All procedures for animal experiments were approved by the Ethics Committee of Beidahuang Group General Hospital and performed in strict accordance with the principles outlined in the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines [19].

Histology analysis

The aortic root was cryosectioned (6 µm) serially at the beginning of the aortic sinus for histology analysis. The lipid accumulation in the aortic valve region was examined by using Oil Red O staining (Cat. No. C0157S, Beyotime Biotechnology, Shanghai, China) and the collagenous fiber was examined by using Masson’s trichrome staining (Cat. No. C0189S, Beyotime Biotechnology). The positive areas were captured under an inverted microscope and quantified by using Image Pro Plus 6.0.

Immunohistochemistry and immunofluorescence staining

The serially aortic cryosections were fixed with 4% paraformaldehyde for 10 min, followed by reactions in 0.1% TritonX-100 for 20 min and 5% bovine serum albumin for 30 min. For immunohistochemical staining, the aortic sections were incubated with rat monoclonal antibody for macrophages [macrophages/monocytes monoclonal antibody (MOMA2); ab33451, Abcam, Cambridge, UK], with 3,3′-diaminobenzidine (DAB) (DA1010, Solarbio, Beijing, China) used for visualization. For immunofluorescence staining, the sections were incubated with rabbit anti-iNOS mAb (ab178945, Abcam), rabbit anti-Arginase-1 mAb (#93668, Cell Signaling Technology, Beverly, MA, USA), with 4′,6-diamino-2-phenylindole (DAPI) (ID2250, Solarbio) used to stain cell nuclei. The fluorescence was image-captured by using a fluorescence microscope (CX23, Olympus, Tokyo, Japan).

Isolation and treatment of mouse peritoneal macrophages

The mice were placed in a supine position and a small incision was made to expose the peritoneal wall. The peritoneal cavity was flushed with 10 ml cold phosphate-buffered saline (ThermoFisher Scientific, Waltham, MA, USA). The lavage fluid was centrifuged at 400 ×g for 5 min and the cell pellet were resuspended in the RPMI1640 medium (ThermoFisher Scientific). Afterwards, isolated peritoneal macrophages were transfected with the recombinant pcDNA3.1 (−)RNF10-overexpressing vector (oe-RNF10) or empty vector (oe-NC) (GenePharma, Shanghai, China) for 24 h by using lipofectamine 3000 reagents (Invitrogen, Carlsbad, CA, USA) as per the instructions provided by the manufacturer. The peritoneal macrophages were subjected to flow cytometric analysis by using rabbit anti-CD86 mAb (ab242142, Abcam) and rat anti-CD206 mAb antibody (MA5-16872, Invitrogen).

Macrophage cell line culture and treatment

Murine macrophages, RAW264.7 (American Type Culture Collection, Manassas, VA, USA), were harvested in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA) in which 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, and 100 U/ml penicillin A and streptomycin existed. Afterwards, the RAW264.7 cells were transfected with oe-RNF10, oe-NC, PPRE-TK-luc reporter constructs together with RNF10 siRNA, or scramble siRNA for 24 h by using lipofectamine 3000 reagents (L3000150, Invitrogen) and then incubated with 50 mg/l ox-LDL (Sigma-Aldrich St. Louis, MO, USA) for 48 h. The RAW264.7 cells were stained with Oil Red O (Cat. No. C0157S, Beyotime Biotechnology) for 15 min. The positive areas were captured under an inverted microscope and quantified by using Image Pro Plus 6.0.

Quantitative real-time polymerase chain reaction (qRT-PCR)

After extraction of total RNA from the aortic tissues and cells by using the TRIzol reagents (15596018CN, Invitrogen), the cDNA synthesis was conducted by using the PrimeScript RT Reagent kit (RR047Q, Takara, Dalian, China). The following qPCR procedures were performed by using the SYBR Master Mixture (Takara) and the LightCycler 480 II System (Roche Diagnostics, Indianapolis, IN, USA). Table 1 lists the primer sequences. The cycle threshold values were standardized to GADPH and the relative amount of target mRNA were calculated using the 2^-ΔΔCT method.

Table 1. The primer sequences used for the qRT-PCR analysis.

Target	Primer sequence
IL-1β	Sense: 5’-CGTGGACCTTCCAGGATGAG-3’
	Antisense: 5’-CATCTCGGAGCCTGTAGTGC-3’
IL-6	Sense: 5’-CGGCCTTCCCTACTTCACAA-3’
	Antisense: 5’-TTCTGCAAGTGCATCATCGT-3’
iNOS	Sense: 5’-GCTTGCCCCTGGAAGTTTCT-3’
	Antisense: 5’-CCTCACATACTGTGGACGGG-3’
IL-10	Sense: 5’-TAACTGCACCCACTTCCCAG-3’
	Antisense: 5’-AAGGCTTGGCAACCCAAGTA-3’
Arg-1	Sense: 5’-CTTGCGAGACGTAGACCCTG-3’
	Antisense: 5’-CTTCCTTCCCAGCAGGTAGC-3’
RNF10	Sense: 5’-ATTTTAGCAACCAGTCCCGTCG-3’
	Antisense: 5’-CCTCATCCCGTCTTCCACCAT-3’
GAPDH	Sense: 5’-TTAGCACCCCTGGCCAAGG-3’
	Antisense: 5’-CTTACTCCTTGGAGGCCATG-3’

Western blotting

After extraction of total protein from the cells by using lysis buffer (AR0105, Boster Biological Technology, Pleasanton, CA, USA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and transfer onto polyvinylidene fluoride membrane were performed. Protein bands were visualized by incubation with primary antibodies for RNF10 (PA5-30339, Invitrogen) and β-actin (MA1-140, Invitrogen), followed by continuous incubation with secondary antibodies. β-actin was used as a loading control.

Data statistics

Results were obtained from six independent repeats and summarized as mean ± SD. All statistical analyses involving Student’s t-test and one-way analysis of variance (ANOVA) plus Tukey’s post-hoc test were obtained by using GraphPad Prism version 8.0 (GraphPad Software, La Jolla, CA, USA) for Windows, with P<0.05 being statistically significant.

Results

Macrophage-specific depletion of RNF10 deteriorates atherosclerotic lesion in mice

In an effort to verify the relationship between macrophage RNF10 and atherosclerosis, we established RNF10^Mac-KO/ApoE^−/− and RNF10^fl/fl/ApoE^−/− mice. As shown by results of Oil Red O staining (Fig. 1A), the RNF10^Mac-KO/ApoE^−/− mice showed a greater atherosclerotic lesion in the aortic root than the RNF10^fl/fl/ApoE^−/− mice. Results of Masson’s trichome staining also showed an increased collagen deposition in the aortic root of the RNF10^Mac-KO/ApoE^−/− mice compared to the RNF10^fl/fl/ApoE^−/− mice (Fig. 1B). In sections of aortic roots stained with MOMA2 antibody recognizing monocytes/macrophages, MOMA2-positive area was significantly larger in RNF10^Mac-KO/ApoE^−/− mice than the RNF10^fl/fl/ApoE^−/− mice (Fig. 1C). These results suggested that macrophage-specific RNF10 deletion deteriorates HFD-induced atherosclerotic development in ApoE^−/− mice.

Fig. 1.

Macrophage-specific depletion of RNF10 deteriorates atherosclerotic lesion in mice. A, Representative images showing atherosclerotic plaque areas by Oil Red O staining and quantitative analysis in the aortic root of RNF10^Mac-KO/ApoE^−/− and RNF10^fl/fl/ApoE^−/− mice (n=10). B, Representative images showing collagen deposition by Masson’s trichrome staining and quantitative analysis in the aortic root of RNF10^Mac-KO/ApoE^−/− and RNF10^fl/fl/ApoE^−/− mice (n=10). C, Representative images showing MOMA2 (macrophage) expression by immunohistochemistry and quantitative analysis in the aortic root of RNF10^Mac-KO/ApoE^−/− and RNF10^fl/fl/ApoE^−/− mice (n=10). **** P<0.0001 by unpaired t test. RNF10, ring finger protein 10; MOMA2, macrophages/monocytes monoclonal antibody.

Macrophage-specific depletion of RNF10 increases inflammatory response in atherosclerotic mice

Next, we studied the effects of macrophage-specific RNF10 deletion on the production of pro-inflammatory and anti-inflammatory factors. Results of immunofluorescence staining showed a higher ratio of iNOS/Arginase-1 in the sections of aortic roots in the RNF10^Mac-KO/ApoE^−/− mice than the RNF10^fl/fl/ApoE^−/− mice (Fig. 2A). As shown in Fig. 2B, higher mRNA expressions of IL-1β, IL-6, iNOS, lower mRNA expressions of IL-10 and Arginase-1 were determined in aorta tissue extracts of the RNF10^Mac-KO/ApoE^−/− mice than the RNF10^fl/fl/ApoE^−/− mice (Fig. 2B). These results suggest that macrophage-specific depletion of RNF10 increases inflammatory response in the aorta of ApoE^−/− mice.

Fig. 2.

Macrophage-specific depletion of RNF10 increases inflammatory response in atherosclerotic mice. A, Representative images showing immunofluorescence intensity of iNOS (red) and Arginase-1 (green), and the ratio of iNOS/Arginase-1 in the aortic root of RNF10^Mac-KO/ApoE^−/− and RNF10^fl/fl/ApoE^−/− mice (n=10). B, The qRT-PCR detection of RNF10 mRNA, M1- and M2-like macrophage marker genes in the aortic root of RNF10^Mac-KO/ApoE^−/− and RNF10^fl/fl/ApoE^−/− mice (n=10). **** P<0.0001 by unpaired t test. RNF10, ring finger protein 10; qRT-PCR, quantitative real-time polymerase chain reaction.

Macrophage-specific depletion of RNF10 affects M1/M2 macrophage status in atherosclerotic mice

In order to confirm the effects of macrophage-specific RNF10 deletion on M1/M2 macrophage status, we isolated peritoneal macrophages from RNF10^Mac-KO/ApoE^−/− and RNF10^fl/fl/ApoE^−/− mice. The RNF10^Mac-KO/ApoE^−/− mice were demonstrated with higher expressions of M1-like macrophage marker genes with lower expressions of M2-like macrophage marker genes in the peritoneal macrophages compared to the RNF10^fl/fl/ApoE^−/− mice (Fig. 3A). Next, a rescue experiment by complementarily overexpressing RNF10 was performed in the peritoneal macrophages from RNF10^Mac-KO/ApoE^−/− mice. As shown by Fig. 3B, RNF10 overexpression was found to decline the expressions of M1-like macrophage marker genes but increase the expressions of M2-like macrophage marker genes in the peritoneal macrophages from RNF10^Mac-KO/ApoE^−/− mice (Fig. 3B). The flow cytometry also demonstrated that RNF10 overexpression could reduce CD86 (M1 surface marker) expression and enhance CD206 (M2 surface marker) expression in the peritoneal macrophages from RNF10^Mac-KO/ApoE^−/− mice (Fig. 3C). These results suggest macrophage-specific depletion of RNF10 promotes macrophage M1 polarization in atherosclerotic mice.

Fig. 3.

Macrophage-specific depletion of RNF10 affects M1/M2 macrophage status in atherosclerotic mice. A, The qRT-PCR detection of M1- and M2-like macrophage marker genes in the peritoneal macrophages from RNF10^Mac-KO/ApoE^−/− and RNF10^fl/fl/ApoE^−/− mice (n=3 biological replicates); **** P<0.0001 by unpaired t test. B, The qRT-PCR detection of RNF10 mRNA, M1- and M2-like macrophage marker genes in RNF10^Mac-KO/ApoE^−/− macrophages after RNF10 overexpression (n=3 biological replicates); **** P<0.0001 by unpaired t test. C, M1/M2 macrophage status detected by the flow cytometry using anti-CD86 and anti-206 antibodies in RNF10^Mac-KO/ApoE^−/− macrophages after RNF10 overexpression; **** P<0.0001 by one-way ANOVA. RNF10, ring finger protein 10; qRT-PCR, quantitative real-time polymerase chain reaction.

RNF10 overexpression antagonizes foam cell formation

With results from animal experiments unveiling on the atheroprotective role of RNF10 in macrophages, we continued to study the regulation of RNF10 in foam cell formation. To this end, we manipulated the expression of RNF10 in RAW264.7 cells by transfecting oe-RNF10 or RNF10 siRNA. Results of qRT-PCR confirmed the forced expression of RNF10 and silencing of RNF10 (Fig. 4A). RNF10 overexpression reduced the accumulation of lipid in ox-LDL-induced foam cells, as shown by the reduced Oil Red O staining in oe-RNF10 compared to oe-NC. In contrast, RNF10 silencing led to a greater Oil Red O staining than its control (Fig. 4B). These results suggest RNF10 overexpression antagonizes foam cell formation.

Fig. 4.

RNF10 overexpression antagonizes foam cell formation in ox-LDL-induced RAW264.7 cells. A, The qRT-PCR detection of RNF10 mRNA expressions in RAW264.7 cells after transfection and ox-LDL stimulation (n=3 biological replicates). B, oe-RNF10, RNF10 siRNA and their negative controls transfected macrophages were treated with 50 mg/l ox-LDL, and the lipid accumulation in the cells was determined using Oil Red O staining after 48 h of incubation (n=3 biological replicates). ** P<0.01 and **** P<0.0001 by unpaired t test. RNF10, ring finger protein 10; ox-LDL, oxidized low-density lipoprotein; qRT-PCR, quantitative real-time polymerase chain reaction.

Discussion

The present study demonstrated RNF10^Mac-KO effectively augmented atherosclerotic lesion and presented more pro-inflammatory M1-like macrophages in ApoE^−/− mice, suggesting the atherosclerosis protective role of macrophage RNF10.

Earlier work has shown that RNF10 downregulation are associated with vascular smooth muscle cell hyperproliferation thus inducing vascular remodeling and vascular restenosis [12, 20]. A recent study [17] reporting the casual relationship between reduced RNF10 in macrophage and age-related inflammation promoted us to test whether RNF10 exerts preventive effects in inflammatory diseases, such as atherosclerosis. Our results showed that RNF10 is reduced in infiltrated macrophages in atherosclerotic lesions of HFD-fed RNF10^Mac-KO/ApoE^−/− mice. This reduced expression of RNF10 in macrophages contributed to production of pro-inflammatory factors, lipid and cholesterol loading, which subsequently promotes atherosclerosis development. In vitro assays also revealed that RNF10 overexpression rectified the changes of M1 and M2 genes in the lesional macrophages. The inflammatory response has implicated during the life cycle of atherosclerosis from atherosclerotic inception to plaque disruption [21]. In the context of atherosclerosis, M1-like macrophages produce proinflammatory factors, such as IL-1β, IL-6, and iNOS, that in turn recruit more inflammatory cells into the lesion area [22]. These inflammatory cells, neutrophils, monocytes, and macrophages, facilitate plaque formation and make plaque more vulnerable [23]. Ubiquitination is a post-translational protein modification that has been recognized as a critical contributor of cellular processes involving vascular inflammation, lipid metabolism and plaque stability in atherosclerosis [24]. Targeting ubiquitin E3 ligases has the chance to prevent atherosclerosis progression [25]. In particular, RNF10 as an E3 ubiquitin ligase inhibited the phosphorylation of p65 and IRF3 and prevented the recruitment of inflammatory cells [17]. In cardiac inflammation, overexpressed RNF10 was found to decline monocyte chemoattractant protein-1, IL-6, and IL-1β [16].

In conclusion, our study demonstrates the atherosclerotic protection of macrophage RNF10in vivo and in vitro. Forced expression of RNF10 may prevent M1-like macrophage infiltration and reduce secretion of proinflammatory factors during atherosclerosis. The present work reveals RNF10 deficiency in macrophage may be contribute to the development of atherosclerosis and RNF10 may be a new opportunity to develop anti-atherosclerotic therapies.

Ethics Approval and Consent to Participate

All procedures for animal experiments were approved by the Ethics Committee of Beidahuang Group General Hospital and performed in strict accordance with the principles outlined in the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.

Consent for Publication

Not applicable.

Competing Interests

No conflict of interest is declared by the authors.

Funding

No funds were received.

Authors’ Contributions

ZKX conceived the study, did the methodology, and wrote the first draft of manuscript, JSX were responsible for data collection and analysis, and LMH were responsible for validation, figure visualization, and revising the manuscript. All authors reviewed and approved the manuscript.

Availability of Data and Materials

Data is provided within the manuscript or supplementary information files.