SFRP4 Reduces Atherosclerosis Plaque Formation in ApoE Deficient Mice

Secreted frizzled related protein 4 (SFRP4), a member of the SFRPs family, contributes to a significant function in metabolic and cardiovascular diseases. However, there is not enough evidence to prove the antiatherosclerosis effect of SFRP4 in ApoE knock-out (KO) mice. ApoE KO mice were fed a western diet and injected adenovirus (Ad)-SFRP4 through the tail vein for 12 weeks. Contrasted with the control cohort, the area of atherosclerotic plaque in ApoE KO mice overexpressing SFRP4 was reduced significantly. Plasma high-density lipoprotein cholesterol was elevated in the Ad-SFRP4 group. RNA sequence analysis indicated that there were 96 differentially expressed genes enriched in 10 signaling pathways in the mRNA profile of aortic atherosclerosis lesions. The analysis data also revealed the expression of a number of genes linked to metabolism, organism system, and human disease. In summary, our data demonstrates that SFRP4 could play an important role in improving atherosclerotic plaque formation in the aorta.


Introduction
Cardiovascular disease (CVD) is the main cause of death in the world, causing 16.7 million deaths every year [1]. Most of CVD are caused by atherosclerosis, which is a disease characterized by the formation of plaque containing lipids and (immune) cells in the intima of large and medium-sized arteries [2]. In the process of atherosclerotic plaque formation, unstable atherosclerotic plaque rupture, vascular stenosis or occlusion caused by platelet aggregation, and thrombosis lead to acute CVD [3]. Atherosclerosis-related infammation is mediated by proinfammatory cytokines, infammatory signaling pathways, bioactive lipids, and adhesion molecules [4]. Te most devastating consequences of atherosclerosis, such as heart attacks and strokes, are caused by superimposed thrombosis [5]. Terefore, atherosclerosis will be a more benign disease if we detect plaque prone to thrombosis and avoid thrombosis [6]. Terefore, it is necessary to further study and develop efective antiatherosclerosis and cardiovascular treatment strategies.
In 1997, Rattner et al. identifed and described a new mammalian gene family, which encodes a secretory protein homologous to the cysteine-rich ligand binding region in the transmembrane receptor frizzled (Fz) family and is named secretory frizzled related proteins (SFRPs), which mediate the cell-cell signal connection of the Wnt signaling pathway [7]. Te size of the SFRPs family proteins is about 30 kDa [8]. Each protein contains a signal peptide sequence, a coiled cysteine-rich domain (CRD), and a conserved hydrophilic carboxyl terminal domain [9]. Based on the structural characteristics of SFRP's protein, it not only combines with Wnt's protein to function but also antagonizes the activity of Wnt protein [7]. On the other hand, it can also bind with the FZ receptor and afect the direction of intracellular signal transduction [7]. As a member of the SFRPs family, since the discovery of SFRP4 [10], studies on its role in diabetes [11], obesity [12], and lipid metabolism [13][14][15][16] have continuously revealed its important roles, providing a new direction and strategy for the treatment of diabetes and lipid metabolism-related diseases. In addition, the latest research studies found that SFRP4 also plays an important role in the occurrence and development of cardiovascular diseases [17]. However, the exact molecular mechanism of its action and signal pathway activation still need to be further studied.
Based on these fndings, this study aims to explore the underlying molecular mechanisms governing SFRP4 regulation of atherosclerosis. In order to confrm this assumption, ApoE knockout (KO) mice, the most popular animal model for human atherosclerosis, were fedwith a western diet and injected with an adenovirus (Ad)-SFRP4 or an Adgreen fuorescent protein (GFP) adenovirus through the tail vein for 12 weeks. After 12 weeks, lipid profles, aortic atherosclerosis, and RNA sequence analysis of diferential gene expression were evaluated in the aorta of the Ad-SFRP4 injected mice and their control counterparts (Ad-GFP injected). Tis study answers two main questions: (1) Does SFRP4 injection through the tail vein afect aortic atherosclerosis and plasma lipids? (2) If not, what is the associated molecular mechanism? Te fndings of this study demonstrate that overexpression of SFRP4 signifcantly inhibits aortic atherosclerosis through multiple signaling pathways except for infammation and oxidative [18].

Animals and Diets.
Eight-weeks-old male ApoE KO mice and wild-type C57BL/6J mice were obtained from the Vital River Company (Vital River Company, Beijing, China). In the experiment, 1 × 10 10 plaque-forming units of Ad-SFRP4 or Ad-GFP (as a control) were introduced into the ApoE KO mice by injection at the tail vein. Pentobarbital sodium (150 mg/kg body weight) was injected intraperitoneally to euthanize the mice. All mice were nourished utilizing a western diet that contained 21% fat and 0.15% cholesterol. Te diets for the mice were produced by Vital River Company (Vital River Company, Beijing, China). Te mice were divided into two cohorts, each comprising ffteen animals. Te mice were housed in an air-conditioned room for a cycle of 12 hours of light and 12 hours dark. Water and food were allowed ad labium. Approval of the animal experiment protocol was obtained from the Laboratory Animal Administration Committee of Xi'an Jiaotong University Health Science Center and performed as per the guidelines for Animal Experimentation of Xi'an Jiaotong University Health Science Center as well as the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication number 85-23, revised 2011).

Construction of the Adenoviral SFRP4 Vector and
Infection of the HEK293 Cells. A recombinant adenoviral vector encoding SFRP4 (Ad-SFRP4) was constructed according to a previously published method [19,20]. SFRP4 cDNA was subcloned into the adenoviral shuttle plasmid pAdTrack-CMV. Following sequence confrmation, the recombinant shuttle plasmid was transformed into the BJ5183 competent cell. Te recombinant adenovirus was packaged and amplifed in HEK293A cells. Following purifcation, the viral titer was detected by TCID50. An empty adenoviral vector (Ad-GFP) was constructed as a control.

Biochemical Analyses.
Mice were fasted overnight, after which blood was drawn from the tail vein. Te blood was mixed with EDTA and then centrifuged at 1,500 rpm for 10 min and a temperature of 4°C to get plasma. Highdensity lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and plasma total cholesterol (TC) were analyzed utilizing commercial assay kits (BioSino Bio-Technology & Science Inc., Beijing, China) [21].
Te plasma sample from each animal was examined in triplicate and measured as per the protocol of the manufacturer utilizing a Benchmark microplate reader (170-6750XTU, Bio-Rad, Veenendaal, Netherlands).

Quantifcation of Atherosclerotic Lesion.
To quantify atherosclerosis, pentobarbital sodium (150 mg/kg) was injected intraperitoneally to euthanize the mice, and the aortic trees were opened up and stained using oil red O. Analysis of the en face lesion size was performed utilizing the image analysis system (WinRoof Mitani Co., Tokyo, Japan) [21,22].
To perform a microscopic examination of atherosclerotic lesions, frozen cross-sections were incised at the level of the aortic root. Specifcally, the analysis involved ten crosssections from each mouse. Te sections were then stained using oil red O and hematoxylin-eosin (H&E) in order to quantify the lesion area. Te image analysis system (Win-Roof Mitani Co., Tokyo, Japan) was employed to quantify the area stained with oil red O [23].

Extraction of Total RNA and Construction of cDNA
Library. Te extraction of total RNA from the aortas was conducted utilizing RNAzol (Takara, Tokyo, Japan). Nanodrop (Termo, Rockford, IL, USA) was utilized to determine the RNA purity and concentration, while the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was utilized to verify integrity. Purifcation of mRNA from the total RNA was performed utilizing the NEBNext ® Poly(A) mRNA magnetic isolation module. Te library with an insert size of 400 bp was built utilizing a NEBNext UltraTM RNA Library Prep Kit adhering to the recommendation of the Illumina manufacturer [24]. Te quality of the library was analyzed using the Agilent Bioanalyzer 2100 system. Te index-coded samples were then clustered on a cBot Cluster Generation System utilizing the TruSeq PE Cluster Kit v4-cBot-HS (Illumina) [25]. Te sequencing of the library was conducted on an Illumina HiSeq. 2500 platform utilizing 100 bp paired end reads (Illumina, San Diego, CA, USA).

Analysis of Diferentially Expressed Genes (DEGs).
Te Perl script was employed for trimming the reads with contaminated adapters, over 0.25% low-quality bases (Phred quality score <20), or over 10% Ns. Subsequently, alignment of clean reads with the mouse reference genome (GRCm38) was conducted utilizing TopHat. Quantifcation and normalization of gene expression were conducted by Cufinks in RPKM (reads per million per kilo bases) [26]. Te DESeq software was utilized to analyze DEGs through a comparison between the Ad-GFP and Ad-SFRP4 cohorts [27]. Te false discovery rate (FDR) was employed to set the signifcant threshold for the p-value in various tests. Te absolute values of FDR <0.05 and fold change ≥2 were used to determine the signifcance of gene expression. To perform pathway and functional enrichment analysis, the DEGs were charted into the Kyoto Encyclopedia of Genes and Genomes (KEGG) datasets, and a p-value of ≤0.05 was utilized to determine the signifcantly enriched KEGG terms.

qRT-PCR Analysis.
qRT-PCRwas performed as previously described [14,20]. Briefy, total RNA was isolated from the aortas of the ApoE KO mice by using the TRIzol Plus (Invitrogen, Carlsbad, CA, USA), and a SuperScript ® III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) was utilized to synthesize cDNA. Ten, the TaKaRa TP800 (TaKaRa Biology Inc., Shiga, Japan) was used to perform real-time PCR analysis. Te fold-change in relative gene expression was calculated using the 2 −ΔΔCT method, using β-actin (for mRNAs) as internal controls. Te sequence of PCR primers is illustrated in Table S1.

Statistical Analysis.
All the data are articulated as mean ± SEM. Statistical analyses were executed utilizing either Welch's t-test if the p-value was not equivalent or student's t-test with an equivalent F-value. Te disparity between the two cohorts is judged to be statistically signifcant if p ≤ 0.05.

Overexpression of SFRP4 in ApoE KO Mice.
To evaluate the protein expression diferences between C57BL/6J and ApoE KO mice, plasma samples were determined by western blotting. It was demonstrated that SFRP4 protein expression was elevated in ApoE KO mice compared to wild type mice (Figure 1(a)). To determine if administering exogenous mouse SFRP4 has an efect on the formation of the atherosclerotic lesion, eight-weeks-old ApoE KO mice were treated systemically with adenoviral vectors expressing mouse SFRP4 (Ad-SFRP4) or control Ad-GFP. Circulating SFRP4 levels were approximately 1.5-fold higher in Ad-SFRP4 compared to Ad-GFP in ApoE KO mice six days after the systemic administration (Figure 1(b)). Te outcomes displayed no signifcant bodyweight or food consumption diferences between Ad-GFP and Ad-SFRP4 injected ApoE KO mice when they reached 20 weeks old ( Figures S1A and S1B).

Te Organ Weight and Plasma Parameters in ApoE KO
Mice. After administrating Ad-SFRP4 for 12 weeks, we evaluated the organ weights of the liver, spleen, kidney, brown adipose tissue, inguinal white adipose tissue, and epididymis white adipose tissue. Tere was no signifcant diference in the organ weight after overexpression of SFRP4 compared to the GFP group ( Figure S2).
No signifcant diferences were observed in metabolic parameters, such as triglyceride, total cholesterol, glucose, and LDL-C in the Ad-SFRP4 group compared to the Ad-GFP of ApoE KO mice (Figures 2(a)-2(c), and 2(e)). However, plasma HDL-C was signifcantly elevated after administered by Ad-SFRP4 compared to the GFP group (Figure 2(d)).

Increased Production of SFRP4 Reduced the Formation of Atherosclerotic Lesions in ApoE KO Mice.
Te size of the en face lesion in the total aorta was reduced considerably by 20% in the Ad-SFRP4 cohort as opposed to the control cohort (Figures 3(a) and 3(b)). Te histological examination illustrated that aortic root atherosclerotic lesions also exhibited a decrease in the Ad-SFRP4 cohort. Microscopic initial lesions in the aortic root shrank considerably by 25% in the Ad-SFRP4 cohort as opposed to the control cohort (Figure 3(c)). Consequently, the lipid area in the lesions stained with oil red O was signifcantly shrunk by 20% in the Ad-SFRP4 cohort (Figure 3(d)).

Overexpression of SFRP4 Induces Genes Expression
Antiatherosclerosis. Since atherosclerotic lesions were considerably smaller in the Ad-SFRP4 injection mice, altered gene expression levels in the lesions were investigated. For comparison, RNA sequence analysis was performed on aorta samples from Ad-SFRP4 and Ad-GFP-injected ApoE KO mice (Figure 4(a)). Te transcriptomic analysis illustrated that there were 97 DEGs in the Ad-SFRP4 mice as opposed to the control mice. Among these DEGs, the upregulated  Table S2).
To investigate their functions, DEGs were grouped into fve categories. Te KEGG pathway analysis ofered more potentially useful information illustrating the pathways pertinent to DEGs in atherogenesis. Based on our DEG outcomes, KEGG pathway analysis illustrated that these DEGs predominantly belonged to organism systems, environmental information, cellular processes, human diseases, and metabolism, such as cholesterol metabolism, chemokine signaling pathway, cytokine-cytokine receptor interaction, and nitrogen metabolism ( Figure 5).

Discussion
Tis study demonstrated that overexpression of SFRP4 inhibited aortic atherosclerosis in ApoE KO mice, however, no signifcant diference in plasma TC, TG, LDL-C, and glucose were observed, but only HDL-C was significantly elevated after overexpression of SFRP4, indicating that the practical efects of SFRP4 is partly dependent on increasing HDL-C plasma cholesterol levels [28]. Tis is a possibility that the achieved maximal efects are partly afected by liver lipid metabolism [29,30]. Aortic lesions were considerably smaller in the SFRP4 overexpressing mice, and this was exemplifed by a decreased accumulation of lipids. Reduced atherosclerotic lesions in the SFRP4 overexpressing cohort could result from multiple possible mechanisms. Hypothetically, the formation of foamy macrophages [31], the adhesion of monocytes to endothelial cells, and the synthesis and release of nitric oxide are vital points to reveal the potential molecular mechanisms contributing to the reduction of atherosclerosis [32,33]. Tis contention was displayed by the outcomes of RNA sequence analysis, which suggested that multiple atherogenic genes, such as cholesterol metabolism, chemokine signaling pathways, and nitrogen metabolism [34][35][36]. Hence, it will be fascinating to examine whether SFRP4 has valuable efects on atherosclerosis. Previous studies have shown that expression of SFRP4 in human ventricular myocardium correlates with apoptosisrelated gene expression [37]. SFRP4 has been detected during cardiovascular maturation and in the adult heart. Moreover, knockdown of SFRP4 attenuates apoptosis to protect against myocardial ischemia/reperfusion injury [17]. Importantly, in our study, we found that the expression of SFRP4 was elevated in ApoE KO mice, indicating that SFRP4 play a role in the progression of atherosclerosis. To verify this hypothesis, we injected Ad-SFRP4 via the tail vein and found SFRP4 overexpression attenuated atherosclerosis plaque formation but also improved the plasma lipids, which was consistent with Zhang and his collegues [18]. In clinical research studies, SFRP4 is associated with impaired glucose and triglyceride metabolism in patients with stable coronary artery disease [38]. Compared to non-CAD patients, human epicardial adipose tissue-derived and circulating SFRP4 levels were increased in patients with CAD, indicating that the level of SFRP4 was independently associated with the presence of CAD [39]. To further confrm the protective efect of SFRP4 overexpression, RNA sequence analysis was performed to evaluate the underlying molecular mechanism about the inhabitation of atherosclerosis. Tese data demonstrated that overexpression of SFRP4 might have the potential to provide protection against atherosclerosis. In this study, RNA sequence analysis of the aorta isolated from ApoE KO mice highlighted the DEGs enriched signaling pathways. SFRP4 is a unique and pleiotropic adipokine that has a protective function against the development of atherosclerosis via several mechanisms [18,40]. Many clinical and epidemiological studies clearly show that HDL-C is negatively correlated with the risk of coronary heart disease (CHD), and it is a key and independent component to predict the risk of CHD [41]. Te elucidation of HDL metabolism has produced therapeutic targets that may increase the level of plasma HDL-C, thereby reducing the risk of CHD [42]. Te concept of reverse cholesterol transport is based on the assumption that HDL has a cardioprotective function, which is a process involving the removal of excess cholesterol accumulated by HDL in peripheral tissues, (such as macrophages in the aorta), transporting it to the liver, and excrete it into the feces through bile. Tereby, overexpression of SFRP4 activated the cholesterol metabolism signaling pathway and promoted the level of plasma HDL-C, which was consistent with the previous study [41]. Typically, Ldlrand Apob-100 were downregulated signifcantly in Ad-SFRP4 group compared to the Ad-GFP group revealed that the formation of foamy macrophages was reduced by the decrease in uptake and transport of LDL [43].
As illustrated in Figure 5, we analyzed the percentage and number of DEGs in these signaling pathways. Te outcomes illustrated that the cholesterol metabolism signaling pathway and cytokine-cytokine receptor interaction were the most extensive functional pathway, accounting for an aggregate of 9 DEGs (∼1% of the total). Existing evidence suggests that these enriched pathways are involved in cholesterol uptake and the ligand-receptor signal response in atherosclerosis [44,45]. Our outcomes illustrated that Ad-SFRP4 mainly afects genes that participate in the formation of foamy macrophages in the aorta. In conclusion, this study presents evidence that overexpression of SFRP4 protects against atherosclerosis. Te underlying mechanism for the protective role of SFRP4 in the progress of plaque formation involves activation of cholesterol metabolism, nitric oxide synthesis, and chemokine signaling pathway. Terefore, circulating overexpression of SFRP4 may ofer a potential efective for preventing atherosclerosis.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon reasonable request.

Ethical Approval
No human studies were carried out by the authors for this article. All institutional and national guidelines for the care and use of laboratory animals were followed and approved by the appropriate institutional committees at the Xi'an Jiaotong University Health Science Center.