Silencing of long non-coding RNA H19 downregulates CTCF to protect against atherosclerosis by upregulating PKD1 expression in ApoE knockout mice

H19 is highly expressed in aortic tissues of ApoE knockout mice with AS

Initially, we conducted RT-qPCR to investigate the expression of H19 in the aortic tissues of normal and AS mice. The results indicated a higher level of H19 in the AS group compared to the control group (p < 0.05) (Figure 1A). Then, FISH assay was employed to explore the subcellular localization of H19, which proved to be present at high concentrations both in nuclei and cytoplasm (Figure 1B). Critical evidence based on examination of human atherosclerosis specimens suggested that H19 was predominantly expressed in the endothelial cell, where its expression was significantly down-regulated in pathological samples compared with healthy carotid artery biopsies [19]. These findings illustrated that elevated H19 in AS aortic tissues might potentially be involved in the pathophysiological process of AS.

Figure 1. High expression of H19 is found in AS aortic tissues. (A) The expression pattern of H19 in normal and AS aortic tissues determined by RT-qPCR. * p < 0.05 vs. the control group. The data were measurement data and expressed by mean ± standard deviation. Data differences between two groups were analyzed by unpaired t-test. n = 6. (B) The subcellular localization of H19 detected by FISH assay (× 400). The experiment was repeated three times independently.

Silencing of H19 inhibits vulnerable plaque formation and intraplaque angiogenesis of ApoE knockout mice with AS

We next conducted RT-qPCR to assess the silencing efficiency of H19 in ApoE knockout mice with AS. In comparison with the NC-ASO group, the level of H19 was significantly lower in aortic tissues of the H19-ASO group (p < 0.05) (Figure 2A). Next, the intimal wall thickening, plaque formation, and plaque vulnerability index scores were evaluated after performing HE staining in order to examine the vulnerable plaque formation following H19 silencing. The NC-ASO group displayed a larger area of atherosclerotic plaques and thinner fibrous caps in addition to enlarged lipid plaque cores. Moreover, a large number of foam cells and deposited cholesterol crystals were observable within the atherosclerotic plaques. Additionally, the inner wall of the artery was thickened however the muscle layer was weakened. The observed atherosclerotic plaque was in an unstable state. A large amount of lipid vacuoles and macrophage infiltration were evident. The smooth muscle layer was thin, with a lack in type I and III collagen fibers. As for the H19-ASO group, we saw a smaller area of atherosclerotic plaques, smooth arterial inner walls and more fibrous caps without sign of fracture. Moreover, no distinct fracture and hemorrhage was evident within the atherosclerotic plaques of these mice. Additionally, a higher quantity of smooth muscle cells and a larger content of type I and III collagen fibers were observed. Moreover, a large number of foam cells accumulated in the atherosclerotic plaques. The cholesterol crystals were asymmetrically distributed, with calcification in some crystals. In general, the atherosclerotic plaques appeared to be in a stable state. The atherosclerotic plaques were less vulnerable, with a lower plaque vulnerability index in the H19-ASO group compared to the NC-ASO group (p < 0.05) (Figure 2B). These results provided ample evidence supporting that silencing of H19 could suppress atherosclerotic vulnerable plaque formation in ApoE knockout mice with AS.

Figure 2. Atherosclerotic vulnerable plaque formation and intraplaque angiogenesis of ApoE knockout mice with AS are inhibited by H19 silencing. (A) The silencing efficiency of H19 assessed by RT-qPCR. * p < 0.05 vs. the NC-ASO group. (B) The atherosclerotic vulnerable plaque formation evaluated by HE staining (× 400) (The arrow referred to lipid vacuoles, * represented inflammatory cells and # indicated fractured smooth muscle.). (C) The number of new blood vessels measured by Immunohistochemical staining (× 400) (The arrow referred to CD34-positive cells). (D) The protein levels of MMP-2, VEGF, p53 and TIMP-1 in atherosclerotic plaques normalized to GAPDH after H19 silencing determined by Western blot analysis. * p < 0.05 vs. the NC-ASO group. The data were measurement data and expressed by mean ± standard deviation. Data differences between two groups were analyzed by unpaired t-test. n = 6. The experiment was repeated three times independently.

To investigate the effect of H19 silencing on intraplaque angiogenesis of ApoE knockout mice with AS, immunohistochemical staining was performed to observe the angiogenesis, the arrow referring to CD34-positive cells. The results illustrated the expression of CD34 in vascular endothelial cells, raising the possibility of their involvement in intraplaque angiogenesis. A large number of newly formed blood vessels were observed in the NC-ASO group, which was significantly lower in the H19-ASO group (p < 0.05) (Figure 2C). Furthermore, Western blot analysis showed significantly decreased protein levels of MMP-2, VEGF and p53 along with an increased protein level of TIMP-1 in the H19-ASO group compared to the NC-ASO group (p < 0.05) (Figure 2D). These obtained results emphasized that H19 silencing repressed intraplaque angiogenesis of ApoE knockout mice with AS.

Silencing of H19 upregulates the level of PKD1

Initially, H19 was overexpressed or knocked down in the atherosclerotic tissues and was evaluated by RT-qPCR. In comparison with the oe-NC group, the expression of H19 was distinctly upregulated in the oe-H19 group, but notably downregulated in the H19-ASO group compared to the NC-ASO group (all p < 0.05) (Figure 3A), thus confirming the successful transfection of oe-H19 and H19-ASO into AS aortic tissues.. RT-qPCR and Western blot analysis showed that in comparison with the oe-NC group, the mRNA and protein level of PKD1 was significantly lower in the oe-H19 group, but markedly elevated in the H19-ASO group compared to the NC-ASO group (all p < 0.05) (Figure 3B, 3C), highlighting that H19 silencing increased the transcriptional as well as the protein level of PKD1 in aortic tissues of AS mice.

Figure 3. The level of PKD1 is elevated by silencing of H19. (A) The silencing or overexpressing efficiency of H19 assessed by RT-qPCR. (B) The transcriptional level of PKD1 after silencing or overexpressing H19 in atherosclerotic tissues determined by RT-qPCR. (C) The protein level of PKD1 after silencing or overexpressing H19 in atherosclerotic tissues normalized to GAPDH evaluated by Western blot analysis (the unprocessed blots are shown in Supplementary Figure 1). * p < 0.05 vs. the oe-NC group; # p < 0.05 vs. the NC-ASO group. The data were measurement data and expressed by mean ± standard deviation. Data differences between two groups were analyzed by unpaired t-test. n = 6. The experiment was repeated three times independently.

H19 inhibits the transcriptional level of PKD1 via binding to CTCF transcription factor

To evaluate the regulatory mechanism of H19 on the transcriptional level of PKD1 and to test whether CTCF was involved in the regulation of PKD1 by H19, we used a RIP assay to assess the binding ability of H19 to CTCF. In comparison with the IgG group, H19 enrichment was significantly upregulated in the CTCF group (p < 0.05) (Figure 4A), suggesting that H19 could potentially bind to CTCF. We next identified the potential binding sites of CTCF on the promoter region of PKD1 using JASPAR (http://jaspar.genereg.net/) (Figure 4B). To further verify the binding relationship between CTCF and PKD1, we adopted the dual-luciferase reporter gene assay. In comparison with the oe-NC group, the luciferase activity of PKD1-Wt was decreased in the oe-CTCF group. However, the luciferase activity of PKD1-Wt was higher in the CTCF-ASO group in comparison with the NC-ASO group (all p < 0.05). There were no significant differences in luciferase activity of PKD1-Mut compared to the different groups (p > 0.05) (Figure 4C). ChIP assay was adopted to further assess the binding ability of CTCF to the promoter region of PKD1, which showed the CTCF enrichment in the promoter region of PKD1 was significantly amplified in the CTCF group in comparison with the IgG group (p < 0.05) (Figure 4D), suggesting that CTCF could successfully bind to the promoter region of PKD1 and that overexpressed H19 induced more binding between CTCF and PKD1.

Figure 4. H19 is capable of suppressing the transcriptional level of PKD1 through binding to CTCF. (A) The binding ability of H19 to CTCF assessed by RIP. *, p < 0.05 vs. IgG. (B) The predicted binding site of CTCF on the promoter region of PKD1 through JASPAR (http://jaspar.genereg.net/). (C) The luciferase activity of PKD1-Wt/Mut after transfection of oe-CTCF or CTCF-ASO determined by dual-luciferase reporter gene assay. * p < 0.05 vs. the oe-NC group; # p < 0.05 vs. the NC-ASO group. (D) The binding ability of CTCF to the promoter region of PKD1 evaluated by ChIP assay. * p < 0.05 vs. IgG. (E) The transfection efficiency of oe-H19 and sh-CTCF determined by RT-qPCR. * p < 0.05 vs. the oe-H19 + sh-NC group. (F, G) The mRNA and protein level of PKD1 normalized to GAPDH after overexpressing H19 or knocking down CTCF measured by RT-qPCR and Western blot analysis (the unprocessed blots are shown in Supplementary Figure 2). * p < 0.05 vs. the oe-H19 + sh-NC group. The data were measurement data and expressed by mean ± standard deviation. Data differences between two groups were analyzed by unpaired t-test; comparisons made among multiple groups were analyzed by one-way ANOVA. n = 6. The experiments were repeated three times independently.

Next, we evaluated the level of PKD1 in cells overexpressing H19 or with knock down of CTCF. RT-qPCR examination of the transfection efficiency of oe-H19 and sh-CTCF (Figure 4E) showed that in comparison with the oe-NC + sh-NC group, the expression of H19 was upregulated in the oe-H19 + sh-NC group and the oe-H19 + sh-CTCF group (p < 0.05). When compared with the oe-H19 + sh-NC group, level of CTCF was downregulated in the oe-H19 + sh-CTCF group (p < 0.05), suggesting that oe-H19 and sh-CTCF were effective. The mRNA and protein level of PKD1 detected by RT-qPCR and Western blot analysis (Figure 4F, 4G) showed that the mRNA and protein level of PKD1 was notably lower in the oe-H19 + sh-NC group compared to the oe-NC + sh-NC group (p < 0.05), but it was salvaged in the oe-H19 + sh-CTCF group (p > 0.05). Therefore, we concluded that H19 suppresses the transcriptional level of PKD1 by binding to CTCF. When H19 was overexpressed, the level of CTCF was unregulated, which would bind to PKD1 and suppress the transcriptional level of PKD1.

H19 participates in atherosclerotic vulnerable plaque formation and intraplaque angiogenesis by down-regulating PKD1 through recruiting CTCF in ApoE knockout mice with AS

Previously, our results demonstrated that silencing of H19 could repress atherosclerotic vulnerable plaque formation and intraplaque angiogenesis of ApoE knockout mice with AS, however the underlying mechanisms remained unknown. Since H19 could inhibit the transcriptional level of PKD1 via binding to CTCF, we proposed a study to investigate whether PKD1 and CTCF were involved in the regulation of vulnerable plaque formation and intraplaque angiogenesis by H19. Initial RT-qPCR results showed that the level of PKD1 was downregulated in the AS group compared to the control group (p < 0.05) (Figure 5A). Subsequently, the ApoE knockout mice with AS were injected with plasmids overexpressing H19, CTCF or PKD1. In comparison with the oe-NC + oe-NC + oe-NC group, the levels of H19 and CTCF were significantly higher in AS aortic tissues of the oe-H19 + oe-CTCF + oe-NC group and the oe-H19 + oe-CTCF + oe-PKD1 group (p < 0.05). In comparison with the oe-H19 + oe-CTCF + oe-NC group, the level of PKD1 was notably elevated in the oe-H19 + oe- CTCF + oe-PKD1 group (p < 0.05) (Figure 5B).

Figure 5. H19 is involved in atherosclerotic vulnerable plaque formation and intraplaque angiogenesis through down-regulating PKD1 by recruiting CTCF in ApoE knockout mice with AS. (A) The expression pattern of PKD1 in the aortic tissues of normal and AS mice determined by RT-qPCR. * p < 0.05 vs. the control group. (B) The overexpressing efficiency of H19, CTCF and PKD1 assessed by RT-qPCR. * p < 0.05 vs. the oe-NC + oe-NC + oe-NC group; # p < 0.05 vs. the oe-H19 + oe-CTCF + oe-NC group. (C) The atherosclerotic vulnerable plaque formation evaluated by HE staining (× 400) (The arrow referred to lipid vacuoles, * represented inflammatory cells and # indicated fractured smooth muscle). (D) The number of new blood vessels measured by Immunohistochemical staining (× 400) (The arrow referred to CD34-positive cells). (E) The protein levels of MMP-2, VEGF, p53 and TIMP-1 in atherosclerotic plaques normalized to GAPDH after transfection determined by Western blot analysis. * p < 0.05 vs. the oe-NC + oe-NC + oe-NC group. The data were measurement data and expressed by mean ± standard deviation. Data differences between two groups were analyzed by unpaired t-test; comparisons made among multiple groups were analyzed by one-way ANOVA. The experiments were repeated three times independently.

HE staining and microscopic examination showed that, in comparison with the oe-H19 + oe-CTCF + oe-NC group, the oe-H19 + oe-CTCF + oe-PKD1 group had a smaller area of atherosclerotic plaques, smooth inner walls of the artery, and more fibrous caps with no fracture. In addition, there was no distinct sign of fracture and hemorrhage within the atherosclerotic plaques, and there were more abundant smooth muscle cells and a larger content of type I and III collagen fiber. Moreover, a large number of foam cells accumulated in the atherosclerotic plaques. The cholesterol crystals were asymmetrically distributed, with calcification in some crystals. In general, the atherosclerotic plaques appeared to be in a stable state. The atherosclerotic plaques were less vulnerable, with a lower plaque vulnerability index (Figure 5C), suggesting that overexpressing PKD1 could partly salvage the vulnerable plaque formation caused by H19.

Immunohistochemical staining showed that CD34 was expressed in the vascular endothelial cells, suggesting its participation in intraplaque angiogenesis. A larger number of newly formed blood vessels were observed in the oe-H19 + oe-CTCF + oe-NC group compared to the oe-NC + oe-NC + oe-NC group (p < 0.05) (Figure 5D). Furthermore, Western blot analysis showed upregulated levels of the angiogenesis-related proteins MMP-2, VEGF and p53 and downregulated level of TIMP-1 in the oe-H19 + oe-CTCF + oe-NC group in comparison to the oe-NC + oe-NC+ oe-NC group (p < 0.05). There were no significant differences in the levels of angiogenesis-related proteins between the oe-H19 + oe-CTCF + oe-PKD1 group and the oe-NC + oe-NC + oe-NC group (Figure 5E). These results revealed the involvement of H19 in atherosclerotic vulnerable plaque formation and intraplaque angiogenesis through down-regulation of PKD1 upon recruitment of CTCF in ApoE knockout mice with AS.

Limitations

We found high expression of lncRNA H19 in mice with AS, while PKD1 was poorly expressed. Furthermore, the mechanistic analyses revealed that lncRNA H19 downregulated PKD1 expression via CTCF. Importantly, H19 promoted atherosclerotic vulnerable plaque formation and angiogenesis by down-regulating PKD1. All experiments were performed on mice, while the H19 expression in serum of patients with AS requires further verification for clinical application. Additionally, the effects of H19 on AS in transcriptome need to be examined by sequencing for an in-depth understanding. Existing literature has demonstrated a high expression of H19 in patients with cardiovascular disease [7, 25, 40, 41]. However, whether the underlying mechanism is in line with mice remains to be further studied.

Future directions

We shall collect and analyze serum from patients with AS and healthy subjects, and perform transcriptome sequencing to retrieve the therapeutic target genes of AS. We shall design in vitro experiments on human cell lines to further unravel the function of the H19/CTCF/PKD1 axis in atherosclerotic vulnerable plaque formation and angiogenesis. In combination with AS-related genes obtained through transcriptome sequencing, the underlying mechanisms in AS will be explored, aiming to support the development of novel therapeutic agents of gene therapy.

Ethic statement

The experimental procedures were conducted with approval of the Institutional Animal Care and Use Committee of Guizhou Provincial People’s Hospital. Mice were treated humanely, and adequate measures were taken to minimize their suffering.

Establishment of AS model in ApoE knockout mice

A total of 132 ApoE knockout mice and 12 C57BL/6J mice (age: 6 - 8 weeks; weight: 20 - 30 g) acquired from the Guangdong Medical Laboratory Animal Center (Guangzhou, Guangdong, China) were chosen as study subjects. These mice were housed in separate cages for 24 weeks with free access to water and food (150 g food for each mouse every day). The 12 C57BL/6J mice were assigned as the control group with normal diet, and ApoE knockout mice were fed a high-fat diet to establish AS model. The high-fat food containing 21% fat and 0.15% cholesterol was processed from conventional food. Then, intermittent repeated smoking was instituted in ApoE knockout mice in order to increase atherosclerotic vulnerable plaque formation.

Animal grouping

After successful establishment of AS models in ApoE knockout mice, the mice were grouped into the AS, negative control (NC)-antisense oligonucleotide (ASO), H19-ASO, oe-NC, oe-H19, oe-NC + sh-NC, oe-H19 + sh-NC, oe-H19 + sh-CTCF, oe-NC + oe-NC + oe-NC, oe-H19 + oe-CTCF + oe-NC, and oe-H19 + oe-CTCF + oe-PKD1 groups, with each group comprising of 12 mice. The following day after grouping, the mice were treated with drugs via an intraperitoneal injection for 13 weeks. The day after completion of the drug treatment regimen, the mice were anaesthetized by an intraperitoneal injection with 4 mL/kg 3% pentobarbital sodium. Next, the chest and the abdomen of mice were excised layer-by-layer using a scalpel and a pair of ophthalmic scissors under aseptic conditions. A section of the separated aorta was resected and fixed conventionally by immersion in 4% paraformaldehyde solution, dehydrated using gradient ethanol, cleared in xylene, and finally embedded with paraffin. A serial of sections of 5 μm thickness was sliced from the aortic root bottom, with 4 identical cross sections for each specimen. After baking at 60°C in an incubator for 6 h, the sections were kept at room temperature. The remaining section of the separated aorta was preserved in liquid nitrogen for further experimentation.

RNA isolation and quantification

Trizol kit was used to extract the total RNA from the tissues, and the miRNeasy Mini Kit (217004, QIAGEN, Hilden, Germany) was used to extract the total RNA from the cells, respectively. Then, RNA was reversely transcribed into complementary DNA (cDNA) in strict accordance with the provided introductions of the PrimeScript RT kit (RR036A, Takara Biotechnology Ltd., Dalian, Liaoning, China). The cDNA was subjected to real time quantitative polymerase chain reaction (qPCR) according to the manufacture’s protocols provided by SYBR^® Premix Ex TaqTM II Kit (RR820A, Takara Biotechnology Ltd., Dalian, Liaoning, China) using an ABI7500 quantitative PCR instrument (7500, ABI Company, Oyster Bay, NY, USA). The primers for H19, CTCF, PKD1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were synthetized by Takara Biotechnology Ltd. (Dalian, Liaoning, China), as shown in Table 1. The 2^-ΔΔCt method was adopted to calculate the relative transcription levels of H19, CTCF, and PKD1 in respect of GAPDH as the internal reference.

Western blot analysis

The total protein in the tissues was extracted using the radio-immunoprecipitation assay lysis buffer (RIPA, R0010, Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) containing phenylmethylsulfonyl fluoride (PMSF), followed by protein quantification. In total, 50 μg of protein was subjected to sodium dodecyl sulfate -polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skimmed milk for 1 h and incubated at 4°C overnight with the following diluted primary antibodies against PKD1 (1 : 1000, bs-2157R), matrix metalloproteinase 2 (MMP-2) (1 : 1000, ab37150), vascular endothelial growth-factor (VEGF) (1 : 1000, ab46154), p53 (1 : 1000, ab131442), tissue inhibitor of metalloproteinases-1 (TIMP-1) (1 : 5000, ab38978), and GAPDH (1 : 5000, ab181602). Except for PKD1, which was purchased from Bioss Biotech (Beijing, China), the other antibodies were purchased from Abcam (Cambridge, MA, USA). After that, the membrane was incubated with the horseradish peroxidase-labeled goat anti-rabbit Immunoglobulin G (IgG) secondary antibody (1 : 1000, HA1003, Huiyan Biotech Co., Ltd., Shanghai, China) at room temperature for 1 h. Finally, membrane development was performed using enhanced chemiluminescence solution (ECL, 808-25, Biomiga, San Diego, CA, USA) for 1 min. The relative expression was expressed as the ratio of the gray value of the target band to that of the internal reference GAPDH band.

Dual-luciferase reporter gene assay

The target gene of CTCF was analyzed and predicted by an online prediction website. The promoter region of PKD1 was introduced into the pGL3-Basic vector (Promega, Madison, WI, USA) to construct the pGL3-PKD1-wild-type (Wt). pGL3-PKD1-mutant (Mut) was also constructed with mutated potential binding sites of CTCF on the promoter of PKD1. The cells were inoculated in the 24-well plate at a density of 3 × 10⁴ cells/well and then transfected with pGL3-PKD1-Wt or pGL3-PKD1-Mut with oe-CTCF and CTCF-ASO as well as the Renilla plasmid. Finally, the luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) with the Renilla luciferase serving as an internal reference. The relative luciferase activity = Relative light unit (RLU) _{firefly luciferase} / RLU _{Renilla luciferase}.

Fluorescence in situ hybridization (FISH) assay

The subcellular localization of H19 in the aorta was identified by FISH assay in accordance with the instructions provided by Ribo^TM lncRNA FISH Probe Mix (Red) (RiboBio Co., Ltd., Guangzhou, Guangdong, China). The paraffin-embedded aorta sections were dewaxed using xylene A and xylene B (each for 10 min), immersed twice in absolute ethanol (5 min each), and detached using protease K at 55°C for 10 min. Subsequently, the sections were rinsed twice with 2 × saline sodium citrate (SSC) (5 min each), dehydrated at -20°C precooled alcohol (70%, 85%, and 100%; 5 min each) and then air-dried. The sections were denatured at 73°C for 5 min, dehydrated using -20°C precooled alcohol of variable concentrations (70%, 85%, and 100%; 5 min each) and air-dried. Then, the hybridization solution was added to the sections, denatured at 73°C for 5 min and further incubated at 37°C overnight. The following day, the sections were washed twice with 48°C preheated 50% formamide or 2 × SSC (5 min/time), rinsed twice using 2 × SSC (5 min/time), and counterstained with 4',6-diamidino-2-phenylindole staining solution for 5 min, followed by 2 × SSC washes (5 min/time). The sections were dried at room temperature in subdued light. Finally, 5 different fields were selected, observed and documented under a fluorescence microscope (Olympus, Tokyo, Japan).

RNA binding protein immunoprecipitation (RIP) assay

The binding of H19 with CTCF was detected using the RIP kit (Millipore, Bedford, MA, USA). The tissues were rinsed using pre-cooled phosphate buffered saline (PBS) three times and homogenized. The tissues were then centrifuged at 1500 rpm for 5 min at 4°C and the supernatant was discarded, while the cells were acquired and further lysed using the RIPA lysis buffer (P0013B, Beyotime Biotechnology Co., Shanghai, China), immersed in an ice-bath for 5 min, and centrifuged at 14000 rpm for 10 min at 4°C for collection of the supernatant. A portion of the cell lysate was used as Input. Then, 50 μL of magnetic beads was rinsed and resuspended in 100 μL RIP wash buffer for each co-precipitation reaction system, and then incubated with 5 μg antibody against CTCF (5 μg/mg of lysate, ab70303) or IgG (1 : 100, ab172730) for co-precipitation. The magnetic bead-antibody complex was rinsed, resuspended in 900 μL RIP wash buffer and incubated with 100 μL cell extract at 4 °C overnight. The samples were placed on a magnetic base to collect the magnetic bead-protein complexes. Subsequently, the precipitated samples and inputs were detached using proteinase K to extract the RNA. H19 expression was detected by performing PCR. The antibodies utilized for RIP assay were CTCF (5 μg/mg of lysate, ab70303) and IgG (1 : 100, ab172730) as NC.

Chromatin immunoprecipitation (ChIP) assay

The fresh frozen tissues were cut into 1 - 3 mm² sections and cross-linked using 1% formaldehyde at room temperature for 15 - 20 min, followed by the addition of 2.5 M glycine to a final concentration of 0.125 M for termination of cross-linking. The sample was sonicated and diluted using 1.8 mL ChIP dilution buffer containing 1 mM PMSF to a final volume of 2 mL. A total of 20 μL sample was taken as Input. The remaining 2 mL sample was incubated with 70 μL of protein A + G agarose/salmon sperm DNA (about 35 μL of precipitate and 35 μL of liquid) at 4°C for 30 min. After centrifugation at 1000 × g for 1 min at 4°C, the supernatant was incubated with the CTCF antibody overnight. The following day, the sample was incubated with the magnetic beads, washed using washing solution and de-cross linked with 5 mmol/L NaCl. The DNA was recovered and detected using reverse transcription (RT)-qPCR with primers specific for PKD1 promoter.

Hematoxylin-eosin (HE) staining

The paraffin-embedded sections were routinely dewaxed and hydrated. Then, the sections were dewaxed using xylene I and II (each for 10 min), and rehydrated in gradient ethanol (100%, 95%, 80%, and 70%, 2 min for each concentration),. After hydration, the sections were conventionally stained with HE (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China), then dehydrated using gradient alcohol, cleared using xylene, and sealed with neutral gum. The aortic plaque formation in mice aortic root was observed under an optical microscope (XP-330, Shanghai Bingyu Optical Instrument Co., Ltd., Shanghai, China).

Evaluation of plaque stability: the pathological sections of mice were processed under HE staining, and the extracellular lipid components (mainly cholesterol crystals and cholesteryl ester) within the aortic plaques of mice in each group were observed under an optical microscope. The sections were further processed with modified movat pentachrome staining, and the smooth muscle cells, foam cells, collagen components and fibrous cap burial in the atherosclerotic plaque of each group were observed under a light microscope. Using of the Medical Image Analysis System, the ratio of extracellular lipids, foam cells, collagen components, and smooth muscle cells to the area of atherosclerotic plaque was calculated according to the following formula: plaque vulnerability index = (extracellular lipid components + foam cells) / (smooth muscle cells + collagen fibers). Observation of the histological pictures was performed in a double-blind manner.

Detection of microvessel density (MVD)

The paraffin-embedded sections were routinely dewaxed and hydrated. Then, the sections were dewaxed with xylene I and II (each for 10 min) and hydrated in gradient ethanol (100%, 95%, 80%, and 70%, 2 min/concentration). Next, the sections were immersed in 3% H₂O₂ for 10 min, treated with high-pressure antigen retrieval for 90 s, and cooled at room temperature. After that, the sections were blocked with 5% bovine serum albumin solution for 30 min at 37°C, followed by overnight incubation at 4 °C with 50 μL of the polyclonal antibody rabbit anti-mouse CD34 (1 : 4000, ab81289, Abcam, Cambridge, MA, USA) or PBS as NC. Subsequently, the sections were further incubated with 50 μL biotinylated goat anti-rabbit IgG (1 : 100, ab172730, Abcam, Cambridge, MA, USA) for 30 min at 37°C. Next, the sections were colored using diaminobenzidine and counterstained with hematoxylin for 5 min, followed by microscopic observation. The criteria used to assess the results of immunohistochemical staining was as follows [42]: the positive cells were reflected as those with a staining degree of higher than 25% and the predominant localization of the staining area in the cytoplasm or cell membrane and vascular endothelium, which was brownish yellow in color. Counting of MVD was based on the number of CD34 positive cells in the field of view. The tan or brownish yellow stained endothelial cell cluster or a single endothelial cell, with a clear demarcation from the adjacent cells, microvessels, and surrounding connective tissue, was counted as a microvessel. If no connections between the structures were evident, their branched anatomical structure could be regarded as a blood vessel. With five high randomly magnification fields selected, the positive expression of CD34 and MVD was determined. Observation of the histological pictures was performed in a double-blind manner.

Statistical analysis

Statistical analysis was conducted using the SPSS 21.0 software (IBM Corp, Armonk, N.Y., USA). Measurement data were presented as mean ± standard deviation, and the enumeration data were expressed as percentage or ratio and tested by χ². The normal distribution of data was tested by the Kolmogorov-Smirnov method. If the data followed normal distribution, comparisons between multiple groups were analyzed by one-way analysis of variance (ANOVA), followed by the Tukey post hoc test. If the data showed a skew distribution, comparisons between multiple groups were analyzed by the Kruskal-Wallis test, followed by Dunn's post hoc test. A value of p < 0.05 was statistically significant.