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

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-specic 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, immuno�uorescence and Western Blot. The results showed that the expression of LKB1 mRNA and protein in arterial tissue of mice increased signi�cantly after overexpression of LKB1. The degree of atherosclerosis, smooth muscle �ber proliferation and lipid accumulation were signi�cantly 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 signi�cantly decreased in the overexpression group (P<0.05). The Immuno�uorescence 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.


Introduction
Atherosclerosis is a chronic in ammatory 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][5][6][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 con rmed 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 calci cation, etc [18][19][20][21][22][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.

Construction of virus vector
The target genes(EnSM22 -mStk11-P2A-EGFP and EnSM22 .EGFP) and adeno-associated virus 9(AAVs9) were purchased from Saiye Biotechnology Co., Ltd.All target genes were knocked into EnSM22 , a VSMCs-speci c 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 identi ed by enzyme digestion, and the positive clones were identi ed 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 puri cation and concentration, the titer of the virus was≥1E+13GC/mL determined by quantitative polymerase chain reaction.

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±1.3g. 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-EnSM22 -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 sacri ced and the aortic tissues were collected for follow-up experiments..

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).

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

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 (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.

Western Blot
Protein was extracted from arterial tissue with RIPA lysate and then quanti ed 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 rst 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.

Immuno uorescence
The para n-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 rst 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.

Statistical analysis
All the experimental data were analyzed by SPSS25.0 statistical software.The normality of the data was tested rst, 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 signi cant.

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 signi cantly 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 signi cantly 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 signi cant difference in blood lipid levels between the two groups before and after the experiment(Figure 1D-E).At the same time,there was no signi cant difference in body weight growth between the two groups during the experiment(Figure 1F).
3.2 AAV9-LKB1 signi cantly 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 signi cantly 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 signi cantly ameliorated in the overexpression group, as the proliferation of the arterial smooth muscle bers and collagen bers were downregulated, the vascular lumen was enlarged, the injury and thickness of intima were both reduced (Figure 2E-F).

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 signi cantly 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 immuno uorescence.The results showed that overexpression of LKB1 signi cantly decreased the in ltration 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 coe cient(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 signi cant (P < 0.01)(Figure 4).

Discussion
Atherosclerosis is a chronic in ammatory 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 di cult 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 nd a target gene that can directly participate in the regulation of atherosclerosis.

The relationship between LKB1 and Cardiovascular Diseases
With the in-depth study of LKB1, it has been con rmed 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 signi cantly 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 bers in arterial tissue decreased signi cantly 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 adenoassociated virus serotype and knock into the vector vascular smooth muscle cell-speci c promoter to selectively overexpress LKB1 in vascular smooth muscle cells.The results showed that there was no signi cant 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 rst described in 1980 [29].Traditional research methods rely on the detection of speci c markers to identify VSMCs phenotypes.Among them, α-SMA encoded by ACTA2 gene is the rst 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 speci c marker protein [30,31].
In addition to speci c 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 con rmed that OPN can promote cell adhesion, proliferation, migration and tissue repair in in ammatory reaction, which is a biomarker of in ammatory diseases such as multiple arteriosclerosis and coronary artery disease [32].It has also been found that OPN is one of the in ammatory 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 identi cation of various in ammatory 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 upregulation 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 immuno uorescence to analyze the co-expression of CD68 and α-SMA.The results showed that the coexpression 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.

Conclusion
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.

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