miR‐145 attenuates phenotypic transformation of aortic vascular smooth muscle cells to prevent aortic dissection

Abstract Background miR‐145 is closely related to vascular smooth muscle cells (VSMC) phenotype transformation; however, the regulatory mechanisms through which miR‐145 regulates the VSMC phenotype transformation under mechanical stretching are unclear. In this study, we evaluated the roles of miR‐145 in VSMCs subjected to mechanical stretching in aortic dissection (AD). Methods The expression of miR‐145 in the aortic vessel wall of model animals and patients with AD was analyzed by quantitative polymerase chain reaction. miR‐145‐related protein‐protein interaction networks and Wikipathways were used to analyze VSMC phenotypic transformation pathways regulated by miR‐145. We used gain‐ and loss‐of‐function studies to evaluate the effects of miR‐145 on VSMC differentiation under mechanical stretch induction and assessed whether Krüppel‐like factor 4 (KLF4) was regulated by miR‐145 in the aorta under mechanical stretch conditions. Results miR‐145 was abundantly expressed in the walls of the normal human aorta, but was significantly downregulated in animal models and the walls of patients with dissection. We found that contractile phenotype‐related proteins were downregulated in VSMCs subjected to mechanical stretching, whereas the expression of secreted phenotype‐related proteins increased. miR‐145 overexpression also downregulated contractile phenotype‐related proteins in VSMCs and suppressed upregulation of phenotype‐related proteins. Finally, under mechanical stretching, KLF4 expression was significantly increased in VSMCs, and overexpression of miR‐145 blocked this effect. Conclusion Our results confirmed that mechanical stretch‐induced phenotypic transformation of VSMCs to promote AD via upregulation of KLF4; this mechanism was regulated by miR‐145, which directly modulated KLF4 expression and VSMC differentiation.


| INTRODUC TI ON
Aortic dissection (AD) refers to tearing of the intima of the aorta, entry of blood into the middle layer of the artery wall, and peeling off of the intima to form a false cavity. Once rupture occurs, massive bleeding and death may result. Thus, AD is a catastrophic disease. [1][2][3] However, the specific pathogenic mechanisms of AD have not yet been reported. VSMCs is closely related to the occurrence and development of AD. [5][6][7] Additionally, microRNAs (miRNAs) have been shown to regulate the phenotypic transformation of VSMCs under physiological and pathological conditions, thereby promoting VSMC differentiation and proliferation. [8][9][10] In particular, miR-145 is closely related to VSMC phenotype transformation 11 ; however, the regulatory mechanisms through which miR-145 regulates VSMC phenotype transformation under mechanical stretching are unclear, and few studies have evaluated VSMCs in patients with AD.
Accordingly, in this study, we collected aortic vessel walls from patients with acute AD and isolated and cultured high-purity aortic VSMCs (AD-VSMCs). We then simulated mechanical stretching caused by hypertension and studies the phenotypes of AD-VSMCs subjected to various stretch conditions. Finally, we investigated the involvement of Krüppel-like factor 4 (KLF4) in miR-145-dependent regulation of the phenotypic transformation of VSMCs in patients with AD under mechanical stretch conditions.

| Bioinformatics analysis of miR-145
The GeneCards database was searched to analyze the position of miR-145 in the chromosome and the distributions of miR-145, miR-221, and miR-21 in different organs and tissues of the human body (https://www.genec ards.org/). miR-145 regulates the proliferation and differentiation of smooth muscle cells by acting on target genes.
We identified 9 target genes involved in these processes using PathCards (https://pathc ards.genec ards.org/). The String tool was used to draw a network diagram of the interaction between miR-145 and its target protein (https://strin g-db.org/). The gene pathway related to the differentiation and proliferation of smooth muscle cells via miR-145 was found using NCBI (https://www.wikip athwa ys.org/). Sites of interaction between miR-145 and KLF4 were analyzed using TargetScan (http://www.targe tscan.org/). Animals were randomly allocated to the experimental group (n = 25) or the control group (n = 10). β-aminopropionitrile (BAPN) was dissolved in 2 ml drinking water for administration at 0.1 g/kg/ day and administered by gavage once a day for 4 weeks. When rats were 7 weeks old, they were injected intraperitoneally with 1.44 mg/ kg AngII dissolved in 0.5 ml, three times a day (at 8-h intervals) for 1 week. The state of the rats was closely observed. If there was a sudden death during the test, the rat was immediately subjected to necropsy using a microscope (CX41; Olympus, Japan) to observe AD formation. If AD was found, the aortic vessels were removed, and the tissues were immediately frozen in liquid nitrogen and stored in a −80°C freezer or fixed in 10% formalin for paraffin embedding.

| Hematoxylin-eosin (HE) staining
Samples had been collected, dehydrated in ethanol solution, and transparentized in dimethylbenzene, which were cut into slices after paraffin embedding. The slices were baked, dewaxed, hydrated, and incubated in hematoxylin solution for 3 min, which were then differentiated in alcoholic hydrochloric acid for 15 s and blued in the Scott bluing to buffer for another 15 s. Next, the slices were washed and incubated in eosin solution for 3 min. Finally, they were dehydrated, transparentized, mounted, and observed under a microscope (CKX41, Olympus, Japan).

| Victoria blue staining
Samples had been collected, dehydrated in ethanol solution, and transparentized in dimethylbenzene, which were cut into slices after paraffin embedding. The slices were incubated in Weigert solution for 10 min, differentiated in alcoholic hydrochloric acid for 10 s, and blued in Victoria blue elastic fiber dyeing buffer for 4 min. By finishing the previous procedures, the slices were K E Y W O R D S aortic dissection, mechanical strain, miR145, phenotype transformation, vascular smooth muscle cells washed and incubated in ponceau S-fuchsin solution for 7 min.
Subsequently, they were washed with weak acid solution and phosphomolybdic acid solution before staining in aniline blue solution for 1 min. Finally, they were dehydrated, transparentized, mounted, and observed under a microscope (CKX41, Olympus, Japan).

| Clinical samples
Patients with acute AD and those who underwent heart transplantation at the Union Hospital of Fujian Medical University

| Extraction, cultivation, and identification of VSMCs from patients with aortic dissection
In patients with acute AD, diseased aortic wall samples were collected, and the intima of the blood vessels was fully peeled off with a scalpel and trimmed into 2 × 2 mm tissue blocks. The tissue blocks were spaced 1 cm apart to cover the bottom of a 25T cell culture flask, and flasks were then placed in an incubator (5% CO 2 , 37°C) for 30 min. An appropriate amount of smooth muscle cell culture medium (SMCM) was added to the culture. The cells migrated out of the tissue block within 5-6 days, and cell growth and fusion reached 90% after 2 weeks. VSMCs were purified using the differential adhesion method. At passages 3-4, cells were showed to have a high purity by immunohistochemistry (α-SMA).

| Cell stretch application
AD-VSMCs were inoculated on Flexcell 6-well plates coated with collagen. When the AD-VSMCs reached 80-90% fusion, serumfree SMCM was added, and cells were cultured for an additional 24 h. The Flexcell 6-well plate was installed on a negative pressure tension plate, the air compressor was applied, and different tensions (0%, 10%, 20%, and 30%) were applied. Briefly, the stretch system controls the negative pressure to make the silicone membrane at the bottom of the stretch plate deform, which generates the corresponding mechanical tension in cells attached to the silicone membrane. Next, AD-VSMCs were collected, total protein was extracted.

| Western blot analysis
RIPA lysis buffer (strong) RIPA (YEASEN, 20101ES60, USA) was used to extract proteins from mechanically stretched and unstretched AD-VSMCs. The protein concentration was determined using the BCA method, and Western blotting was then used to detect protein levels. Briefly, protein extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were then blocked with 5% skim milk for 2 h and incubated with primary antibodies overnight at 4°C. After washing three times with TBST, the membranes were incubated with secondary antibodies for 1 h. Visualization was performed using an enhanced chemiluminescence detection system, and Photoshopcs3 software was used to analyze the relative band

| Real-time quantitative polymerase chain reaction (RT-qPCR)
To detect the expression of miRNAs, including miR-145, we performed RT-qPCR using universal cDNA synthesis and SYBR Green Master Mix (Denmark). U6 rRNA expression was used as an internal control. The specific RNA PCR primer set and U6 primers were obtained from Exiqon (Denmark).
To detect levels of pri-miR-145, total RNA was collected using a PrimeScript RT kit (Takara, Japan) and reverse transcribed into cDNA. Real-time PCR was then performed using SYRB Premix Ex Taq kit (Japan). The PCR cycle conditions were as follows: 95°C for 3 min, 95°C for 40 s, 56°C for 10 s, 40 cycles, and 72°C for 10 s.

| Transfection of AD-VSMCs
The medium was then incubated with serum-free SMCM for 48 h.

| Statistical analysis
Continuous variables were presented as means with SD or medians with IQR, and analyzed by analysis of variance (ANOVA) models or student's t test (two-tailed) or rank-sum test. Statistical analyses were performed using SPSS version 24.0 and a p-value <0.05 was considered statistically significant.  Figure 1D).

| Successful establishment of an AD model
Among the 25 experimental rats, 17 died suddenly after the drug intervention. Necropsies showed that all causes of death were AD, and none of the surviving rats showed AD. The incidence of AD in our animal model was 68%. The blood vessels in SD rats with AD were dissected ( Figure 2A) and stained with hematoxylin and eosin ( Figure 2B and C) and Masson staining ( Figure 2D and E). The structure of the aortic wall in the control group was complete and continuous. In AD rats, the aortic wall contained broken elastic fibers, and blood cells enter the wall, leading to tearing, peeling, and the formation of a false lumen. These results supported that the AD rat model was successfully established.

| miR-145 expression was downregulated in model rats and patients with AD
The GTEx and BioGPS databases from GeneCards showing the miR-145 expression in normal human tissues. For tissues with more than 100 M reads, miR-145 was considered highly expressed ( Figure 4A).
The results showed that miR-145 was abundantly expressed in the arteries, small intestine, colon, stomach, esophagus, bladder, uterus, and prostate ( Figure 4B).
RT-qPCR showed that miR-145 was significantly downregulated in aorta samples from the model rats compared with that in the control group ( Figure 4C). In order to further verify the effects of me-

| Mechanical stretch-induced phenotypic transformation of VSMCs in model rats and patients with AD
From the diseased vascular tissues of patients with AD, we successfully separated tissues and cultured primary VSMCs Compared with control cells, AD-VSMCs subjected to 30% mechanical tension showed decreased expression of VSMC contraction markers, including α-SMA, SM22, and the expression levels of markers of contraction were also low ( Figure 6A and B). In contrast, the expression levels of the VSMC secretion markers PCNA and OPN were higher under mechanical stretching than under static culture.
The expression of secretion marker proteins increased after application of mechanical stretching, and expression levels increased as mechanical tension increased ( Figure 6C and D).  Figure 6E and F).

| miR-145 targeted KLF4 to participate in mechanical stretch-induced phenotypic transformation of VSMCs
Compared with statically cultured AD-VSMCs, after applying mechanical stretch, the expression of KLF4 protein was significantly increased in AD-VSMCs. However, upregulation of KLF4 under mechanical stretching was inhibited by the miR-145 mimic ( Figure 6G and H). Notably, after knockdown of KLF4 by siRNA in AD-VSMCs, KLF4 expression was inhibited. Under mechanical stretching, the expression of the contraction markers of AD-VSMCs was increased compared with that in the control group ( Figure 6I and J).

| DISCUSS ION
Surgical operation is the only approach for curing AD; however, the surgery is invasive and costly, and mortality rates are still high, even after treatment. Therefore, it is necessary to identify biological markers and therapeutic targets in order to improve outcomes in patients with AD.
Hypertension is closely related to the occurrence of AD. Because of the features of blood flow and blood pressure, 12,13 blood vessels are constantly exposed to mechanical forces in the form of shear stress and stretching, and VSMCs begin to differentiate from the contraction type to the secretion type. 14 In summary, our findings showed that KLF4 may be a potential therapeutic target in patients with AD. We also showed that miR-145 inhibited the differentiation of AD-VSMCs by increasing KLF4 degradation or reducing KLF4 expression; these changes delayed the progression of hypertension-induced aortic wall remodeling and further suppressed the occurrence of AD.

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to disclose.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.