LKB1 delays atherosclerosis by inhibiting phenotypic transformation of vascular smooth muscle cells

doi:10.21203/rs.3.rs-2634984/v1

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LKB1 delays atherosclerosis by inhibiting phenotypic transformation of vascular smooth muscle cells

https://doi.org/10.21203/rs.3.rs-2634984/v1

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Background and objective: Although liver kinase B1 (LKB1) is a well-known tumor suppressor gene, and its encoded protein has important biological functions, it is not clear whether LKB1 can inhibit atherosclerosis by regulating vascular smooth muscle cells (VSMCs). The purpose of this study is to explore the relationship among LKB1, VSMCs and atherosclerosis.

Methods and results: ApoE^-/- mice with VSMCs-specific overexpression of LKB1 were constructed by adeno-associated virus transfection technique, and then fed with high-fat diet for eight weeks. The effect of LKB1 overexpression on atherosclerosis in mice was investigated by oil red O staining, HE staining, immunofluorescence and Western Blot. The results showed that the expression of LKB1 mRNA and protein in arterial tissue of mice increased significantly after overexpression of LKB1. The degree of atherosclerosis, smooth muscle fiber proliferation and lipid accumulation were significantly alleviated in the overexpression group. The results of Western Blot showed that the expression of α-SMA was increased, while the expression of OPN and CD68 was significantly decreased in the overexpression group (P<0.05). The Immunofluorescence results of Image Pro Plus software analysis showed that the co-localization relationship between α-SMA and CD68 was more obvious in the control group (P < 0.01).

Conclusion: Our results suggested that LKB1 can delay the progression of atherosclerosis by inhibiting the phenotypic transition of VSMCs.

LKB1

Atherosclerosis

Vascular smooth muscle cells

Phenotypic transformation

Atherosclerosis is a chronic inflammatory lesion involving the intima to media of large and medium-sized arteries[1, 2], which will cause vascular stenosis and occlusion. According to statistics, atherosclerosis is one of the most common causes of cardiovascular and cerebrovascular diseases, and it is also the leading cause of death worldwide[3].

The pathogenesis of atherosclerosis involves a variety of theories. According to the traditional viewpoint, atherosclerosis starts with endothelial dysfunction, and lipid accumulation while foam cell formation run through the development of atherosclerotic [4-7]. In recent years, it has been found that vascular smooth muscle cells(VSMCs), which is the most important cell type in the process of atherosclerotic, participates in the whole process of atherosclerosis through proliferation, migration and differentiation. In 2017, LauraS et al showed that more than 80% of the cells in arterial plaques were derived from VSMCs [12].

LKB1 is a tumor suppressor factor discovered by Hemminki[13], which is a serine / threonine protein kinase that exists widely in organisms. It mainly activates AMPK by phosphorylation and plays an important role in regulating cellular energy metabolism, inhibiting cell growth and maintaining cell polarity[14, 15]. In recent years, more and more studies have proved that LKB1 is also closely related to cardiovascular diseases. At present, it has been confirmed that the absence of LKB1 in the embryonic stage will affect embryonic angiogenesis, resulting in abnormal embryonic development and death [16-17]. In mature individuals, the absence of LKB1 will cause a series of problems, such as myocardial hypertrophy, cardiac dysfunction, vascular endothelial dysfunction, atherosclerosis, aortic dissection, vascular calcification, etc [18-23]. Among them, LKB1 has been shown to inhibit atherosclerosis by inhibiting the formation of macrophage-derived foam cells [12], and inhibiting cholesterol and fatty acid synthesis[24].However, it has not been reported whether LKB1 can participate in the formation of atherosclerosis by regulating VSMCs, so the main purpose of this study is to explore the relationship among LKB1, VSMCs and atherosclerosis.

2.1 Construction of virus vector

The target genes(EnSM22ɑ-mStk11-P2A-EGFP and EnSM22ɑ.EGFP) and adeno-associated virus 9(AAVs9) were purchased from Saiye Biotechnology Co., Ltd. All target genes were knocked into EnSM22ɑ, a VSMCs-specific promoter. The circular plasmid expression vector was digested in water bath at 37 ℃, and the large fragments of the digested plasmid were recovered by 1% agarose gel electrophoresis. The large fragment and the target gene CDS sequence were recovered for gene recombination, and the recombination reaction was carried out using a seamless cloning kit (Novozan Biotechnology Co., Ltd., China). The plasmid was extracted with a small plasmid extraction kit (Tiangen Biotechnology Co., Ltd.), the plasmid was identified by enzyme digestion, and the positive clones were identified by sequencing. The correct recombinant plasmid was used in the next step of cell transfection. The target plasmid and two helper plasmids were co-transfected into HET293T Cells for 72 hours. The supernatant was concentrated with PEG8000, and the virus was collected by repeated freeze-thaw lysis. After purification and concentration, the titer of the virus was≥1E+13GC/mL determined by quantitative polymerase chain reaction.

2.2 Animals and Treatment

The animal experiment was approved by the Animal Committee of Guangdong Pharmaceutical University. Thirty 8-week-old male ApoE^-/- mice were purchased from Saiye Biotechnology Co., Ltd., with each weighing 23.2±1.3 g.

30 mice were randomly divided into control group (n=15) and overexpression group (n=15). The mice in the overexpression group and control group were injected with AAVs9-EnSM22ɑ-mStk11-P2A-EGFP (AAVs9-LKB1) and AAVs9-EnSM22-EGFP (AAVs9-EGFP) through tail vein, and the injection dose of the virus in each mouse was 4×107TU (200ul). All mice received a high-fat diet (protein 22.63%, carbohydrate 45.51%, fat 20.06%) for 8 weeks to induce atherosclerosis. The body weight of mice was measured every Tuesday and Friday. At the end of the 8th week, the mice were sacrificed and the aortic tissues were collected for follow-up experiments..

2.3 Detection of blood lipid level.

The blood lipids were measured at 1st and 8th week, respectively. An Automatic biochemical analyzer was used to detect blood lipid levels, including total cholesterol(TC), triglyceride(TG), low-density lipoprotein cholesterol(LDL-C) and high-density lipoprotein cholesterol(HDL-C).

2.4 Histopathological examination

The aortic tissue was made into paraffin sections and stained with oil red O and hematoxylin-eosin (HE). Then the lipid accumulation and smooth muscle fiber proliferation were analyzed by Image Pro Plus.

2.5 Quantitative PCR

Total RNA was extracted from arterial tissues by TRIzol method, and then the RNA was reversely transcribed into cDNA using Prime Scrip RT reagent Kit (Chinese Plateau Biotechnology). According to the instructions of kit SYBR Prime Script Ex Taq Ⅱ (High altitude Biotechnology, China), the qPCR reaction system was set up to carry out qPCR reaction. With GAPDH as the internal control, the primers used were as follows: the upstream primer of LKB1: 5‘-GGAGGACGAGGACTTGTTTGA-3’, the downstream primer of LKB1: 5‘-GTCCATTCTGACCCACTTCCT-3’, the upstream primer of GAPDH: 5‘-AGAAGGCTGGGGCTCATTTG-3’, the downstream primer of GAPDH: 5‘-AGGGGCCATCCACAGTCTTC-3’. The relative expression of LKB1 was detected by the 2-△△CT method.

2.6 Western Blot

Protein was extracted from arterial tissue with RIPA lysate and then quantified with BCA protein concentration determination kit (Seville Biotechnology Co., China). After fully mixing the protein with the buffer, the protein is denatured by heating. The obtained protein samples were loaded on 10%SDS-PAGE gel for electrophoresis, and then transferred to PVDF membrane. After sealing the PVDF membrane in 5% defatted emulsion at room temperature for 1 h, it was incubated overnight with the first antibody containing anti-LKB1, AMPK, α-SMA, OPN and CD68 at 4℃, and then incubated with appropriate horseradish peroxidase coupled secondary antibody at room temperature for 1 h. Finally, a chemiluminescence kit (Beyotime, China) was used to display protein imprinting. The relative intensity of protein bands was measured by Image J.

2.7 Immunofluorescence

The paraffin-embedded sections were dewaxed and rehydrated, then placed on a pressure cooker with citric acid antigen repair solution, heated to boiling, then reduced to room temperature within 30 minutes and rinsed with PBS. 3% H2O2 solution was dropped into the slices and 10min was incubated at room temperature to eliminate the effect of H2O2. An appropriate amount of sealing solution was added to the slices and 30min was sealed at room temperature. Then the first antibody against α-SMA and CD68 (1:100) was added and incubated overnight at 4 ℃. After 24 hours, the slices were placed at room temperature to reheat 30min, and then the donkey anti-goat secondary antibody labeled with horseradish peroxidase was diluted according to 1:80 and dripped onto the slice. The slices were placed at room temperature to incubate 30min without light. The processed slices were stained with DAB and hematoxylin in turn. Finally, the slices were dehydrated and sealed with neutral resin. The slices were imaged, observed and photographed by the microscope, and the expression of α-SMA and CD68 was analyzed by Image Pro Plus.

2.8 Statistical analysis

All the experimental data were analyzed by SPSS25.0 statistical software.The normality of the data was tested first, and the results were expressed as mean ±standard deviation. Then the homogeneity test of variance is carried out. If the variance is homogeneous, the mean t-test of two independent samples is used, and the t 'test is used if the variance is uneven. When P < 0.05, the difference was statistically significant.

3.1 AAV9-LKB1 increases the expression of LKB1 in arteries

The results of qPCR showed that the mRNA level of LKB1 in the overexpression group was significantly higher than that in the control group(Figure 1A) and the results of WB showed that the expression level of LKB1 protein in artery specimens in the overexpression group was significantly higher than that in the control group (Figure 1B). The above results showed that the expression of LKB1 in VSMCs of mice was successfully increased by tail vein injection of AAV9-LKB1.

The results showed that there was no significant difference in blood lipid levels between the two groups before and after the experiment(Figure 1D-E). At the same time,there was no significant difference in body weight growth between the two groups during the experiment(Figure 1F).

3.2 AAV9-LKB1 significantly reduced the degree of atherosclerosis in ApoE^-/- mice

8 weeks’ of high fat diet feeding resulted in large amount of lipid accumulation and atherosclerosis in ApoE^-/- mice, while oil red O staining showed significantly decreased degree of atherosclerosis and lipid accumulation in the overexpression group (Figure 2A-C). The results of HE staining also showed that the degree of atherosclerosis was significantly ameliorated in the overexpression group, as the proliferation of the arterial smooth muscle fibers and collagen fibers were downregulated, the vascular lumen was enlarged, the injury and thickness of intima were both reduced (Figure 2E-F).

3.3 AAV9-LKB1 inhibited the phenotypic transition of VSMCs.

We used WB to detect the expression levels of osteopontin(OPN), α-SMA and CD68 in arterial specimens. The results showed that the expression level of OPN and CD68 in the overexpression group was significantly lower than that in the control group, while the expression of α-SMA was increased, indicating the inhibition of phenotypic transition of VSMCs after LKB1 overexperssion (Figure 3). At the same time, we analyzed the co-expression of α-SMA and CD68 by immunofluorescence. The results showed that overexpression of LKB1 significantly decreased the infiltration of CD68 positive cells and partially reversed the loss of α-SMA positive cells in the inner layer of blood vessel. (Figure 4). We further used Image Pro Plus to analyze the co-expression of α-SMA and CD68, and the results were expressed by Pearson’s correlation coefficient(PCC). PCC > 0.5 indicates that there is a co-localization relationship between the two target proteins, otherwise there is no co-location relationship. The results showed that there was a certain co-localization relationship between α-SMA and CD68 in the arterial samples of the two groups, but the co-localization relationship of α-SMA and CD68 was more obvious in the control group, and the difference was statistically significant (P < 0.01)(Figure 4).

Atherosclerosis is a chronic inflammatory disease of blood vessels, which is the pathological basis of most cardio-cerebrovascular diseases. At present, the mainstream treatment strategy for atherosclerosis is still the comprehensive treatment based on statins, however, for some high-risk patients, conventional doses of statins cannot reduce the risk of cardiovascular disease. Besides, increasing the dose is not only difficult to obtain a satisfactory effect, but also will increase adverse drug reactions. In addition, some people are intolerant to statins and have a variety of adverse drug reactions, so the traditional comprehensive strategy for the treatment of atherosclerosis has been unable to meet all treatment needs[25, 26]. At present, the biological targeting drug proprotein converting enzyme subtilysin 9 (PCSK9) inhibitor can reduce the level of LDL-C by reducing PCSK9-mediated LDL-R degradation, thus reducing the risk of atherosclerosis [27]. However, from the pathological basis of atherosclerosis, PCSK9 inhibitors mainly play a role in the pathological mechanism of lipid accumulation, but there is no clear regulatory effect on VSMCs throughout the process of atherosclerosis. Therefore, starting from the pathological mechanism of atherosclerosis, it may be more meaningful to try to find a target gene that can directly participate in the regulation of atherosclerosis.

4.1 The relationship between LKB1 and Cardiovascular Diseases

With the in-depth study of LKB1, it has been confirmed that LKB1 is closely related to a large number of cardiovascular diseases. In recent years, attention has been paid to the relationship between LKB1 and atherosclerosis. The study found that the level of LKB1 decreased significantly in both serum and atherosclerotic plaques of patients with coronary heart disease. This suggests that there may be a negative correlation between LKB1 and the degree of atherosclerosis. In our experiment, compared with the control group, the degree of atherosclerosis and lipid accumulation, the proliferation of VSMCs and the secretion of collagen fibers in arterial tissue decreased significantly after overexpression of LKB1, which directly indicated that overexpression of LKB1 could inhibit the occurrence and development of atherosclerosis in mice to some extent.

In order to rule out that LKB1 inhibits atherosclerosis by reducing blood lipids, we use AAVs9 adeno-associated virus serotype and knock into the vector vascular smooth muscle cell-specific promoter to selectively overexpress LKB1 in vascular smooth muscle cells. The results showed that there was no significant difference in body weight and blood lipid levels in the mice fed with high-fat diet for 8 weeks, suggesting that the inhibition of atherosclerosis by LKB1 does not depend on the regulation of blood lipid levels, but directly through the regulation of VSMCs function. The experimental results of Liu et al show that LKB1 can inhibit the formation of foam cells by macrophages in atherosclerosis[12]. As complements, our experiment indicated that LKB1 can also inhibit atherosclerosis by regulating the biological function of VSMCs.

4.2 LKB1 inhibits the development of atherosclerosis by regulating the phenotypic transformation of VSMCs.

Under normal physiological conditions, VSMCs is located in the media of blood vessels and has important biological functions such as constricting blood vessels, regulating vascular tension and maintaining blood pressure. However, under the stimulation of various pathological factors, the biological characteristics of VSMCs will change, such as the enhanced ability of secretion, proliferation and migration, and can be dedifferentiated into various types of cells, including macrophage-like cells, osteoblast-like cells, mesenchymal stem cell-like cells and other cell types, thus participating in the pathophysiology of atherosclerosis [28]. This phenotypic plasticity of VSMCs is called phenotypic transition and was first described in 1980 [29]. Traditional research methods rely on the detection of specific markers to identify VSMCs phenotypes. Among them, α-SMA encoded by ACTA2 gene is the first marker protein expressed during VSMCs differentiation, and it is also the most abundant single protein in contractile VSMCs, accounting for about 40% of the total cell protein, which is closely related to the contractile function of VSMCs and highly selective to VSMCs and VSMCs-like cells. therefore, α-SMA is the most widely used VSMCs specific marker protein[30, 31].

In addition to specific marker proteins, some extracellular matrices are also associated with VSMCs phenotypic transformation, such as OPN. OPN is a secretory multifunctional glycophosphate protein. It has been confirmed that OPN can promote cell adhesion, proliferation, migration and tissue repair in inflammatory reaction, which is a biomarker of inflammatory diseases such as multiple arteriosclerosis and coronary artery disease [32]. It has also been found that OPN is one of the inflammatory mediators in atherosclerosis and is closely related to pathological processes such as macrophages and neutrophil recruitment and migration [33]. Liu et al found that OPN can regulate the proliferation and migration of VSMCs in atherosclerosis with or without αvβ3/MMP-9 pathway [34]. The experiments of Tohru et al also show that the secretion of OPN is related to VSMCs-derived foam cells [35]. Therefore, in atherosclerosis, the expression level of OPN is considered to be related to the phenotypic transition of VSMCs, that is to say, OPN can be used to judge the status of VSMCs to some extent.

In this experiment, we made a comprehensive judgment on the phenotype of VSMCs by detecting the traditional method of α-SMA expression, combined with the determination of OPN expression level. The results showed that compared with the control group, the expression of α-SMA in the arterial specimens of the overexpression group was increased, while the expression of OPN was decreased, which indicated that the proliferation and migration ability of VSMCs was weakened. That is, the overexpression of LKB1 inhibits the proliferation and migration of VSMCs. This is consistent with the results of histopathological examination.

In addition, our experiments also suggest that VSMCs can participate in the pathological process by converting to macrophage-like cells. In the experimental results, oil red O staining showed that the lipid accumulation was serious and the formation of foam cells was increased in the control group, while further Western Blot detection showed that the expression of CD68 in the control group was up-regulated. CD68 is generally regarded as a pedigree marker of macrophages and has an important application in the identification of various inflammatory cells [36, 37]. However, some studies have shown that in the process of phenotypic transformation of VSMCs, some VSMCs can dedifferentiate into macrophage-like cells and express CD68 [12, 38, 39]. Therefore, in our experiment, there are two possibilities for up-regulation of CD68 expression, that is, VSMCs dedifferentiates into macrophage-like cells and expresses CD68 to increase the expression of CD68, or monocyte-macrophage-derived macrophages up-regulate CD68 expression through clone proliferation. In order to further explore the source of CD68, we used immunofluorescence to analyze the co-expression of CD68 and α-SMA. The results showed that the co-expression relationship between CD68 and α-SMA in the control group was more obvious than that in the overexpression group, which indicated that the number of macrophage-like cells dedifferentiated by VSMCs in the control group was increased. The experimental results show that VSMCs can dedifferentiate into macrophage-like cells and participate in the formation of foam cells in the process of atherosclerosis, which is consistent with most of the current research conclusions. Therefore, our experimental results also show that inhibiting the transformation of VSMCs into macrophage-like cells is also one of the mechanisms of LKB1 delaying atherosclerosis.

To sum up, this study showed that VSMCs participates in the pathophysiological process of atherosclerosis through phenotypic transformation, while overexpression of LKB1 can delay the occurrence and development of atherosclerosis by inhibiting the phenotypic transformation of VSMCs, this means that LKB1 may become a more comprehensive genetic target for the treatment of atherosclerosis.

Acknowledgments: This work was supported by the Guangdong Provincial Innovation Team Project of Colleges and Universities (No. 2019KCXTD018) and the Scientific Research Project of Guangdong Bureau of Traditional Chinese Medicine (No. 20221229).

Funding

This work was supported by the Guangdong Provincial Innovation Team Project of Colleges and Universities (No. 2019KCXTD018) and the Scientific Research Project of Guangdong Bureau of Traditional Chinese Medicine (No. 20221229).

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

CKC and RC designed the experimental process, CKC, JQL, CYZ, QZ and Zihao Zhou were responsible for the implementation of the experiment, CKC wrote the manuscript, TDL and RZ were responsible for proofreading the manuscript, and ZHZ supervised all the work.

Ethics approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of The First Affiliated Hospital of Guangdong Pharmaceutical University.

Consent to participate

Not applicable.

Consent to publish

Not applicable.

Code availability

Not applicable.

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LKB1 delays atherosclerosis by inhibiting phenotypic transformation of vascular smooth muscle cells

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1. Introduction

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4. Discussion

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