Int J Clin Exp Med 2019;12(12):13265-13273 www.ijcem.com /ISSN:1940-5901/IJCEM0101150. Original Article Expression of Gax in thoracic aortic aneurysm and its effect on phenotypic transformation of thoracic aortic smooth muscle cells. Hui Zheng, Jidong Liu, Jianggui Shan, Song Xue. Department of Cardiovascular Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China. Received August 19, 2019; Accepted November 5, 2019; Epub December 15, 2019; Published December 30, 2019. Abstract: Objective: To investigate the expression of growth arrest-specific homeobox gene (Gax) in thoracic aortic aneurysm (TAA) and its effect on the phenotypic transformation of thoracic aortic smooth muscle cells (TASMCs). Methods: The thoracic aortic aneurysm wall tissues from 16 patients with degenerative ascending thoracic aortic aneurysm (DATAA) were enrolled in this study. Further, 16 normal control samples from patients with coronary artery disease (CAD) undergoing coronary artery bypass grafting (CABG) during the same period were collected. Real-time PCR and Western blot were used to detect the mRNA and protein expressions of Gax in the tunica media of thoracic aortic aneurysm wall. Digestion and adherence method was used to primarily culture normal human TASMCs and TASMCs from TAA. Immunofluorescence staining was used to quantify the expressions of α-actin and Gax. The TASMCs were transfected with recombinant adenovirus vector Ad-Gax. Western blot assessed the effects of Gax over-expression on the expressions of Calpoin and SM-MHC 11 proteins in TASMCs, CCK-8 detected its ef- fects on the proliferation and Transwell assay detected its effects on the migration of TASMCs. Results: According to Real-time PCR and Western blot, the mRNA and protein expressions of Gax in the tunica media of thoracic aortic aneurysm wall were significantly lower than those in normal aorta. According to immunofluorescence staining, the expressions of Gax and α-actin in TASMCs of patients with TAA were significantly lower than those in normal TASMCs of patients with CAD. The over-expression of Gax up-regulated the expressions of Calpoin and SM-MHC 11 proteins in TASMCs. According to CCK-8 method, the over-expression of Gax significantly inhibited the proliferation of TASMCs. According to Transwell assay, the over-expression of Gax significantly inhibited the migration of TASMCs. Conclusion: The expression of Gax decreases in tunica media and TASMCs from TAA, promoting the transformation of TASMCs from synthetic phenotype to contractile phenotype.. Keywords: Growth arrest-specific homeobox gene, thoracic aortic aneurysm, vascular smooth muscle cells, pheno- typic transformation. Introduction. Thoracic aortic aneurysm (TAA), a clinically common macrovascular disease, mainly refers that pathological changes in aortic wall lead to aortic dilatation and even life-threatening com- plications such as aortic dissection rupture, aortic insufficiency (AI), and heart failure [1, 2]. Epidemiological investigation has shown that the disease has an increasing incidence and becomes more common among young people . According to studies, thoracic aortic smooth muscle cells (TASMCs) are the main cells of tunica media of thoracic aorta, playing an im- portant role in maintaining vascular homeosta-. sis, reducing vascular injury-induced stress re- sponses, and regulating blood pressure [4, 5]. The phenotypic transformation of TASMCs is a main pathological feature of TAA and plays a pivotal role during its pathogenesis [6, 7].. Growth arrest-specific homeobox (Gax) is an inhibitory transcription factor widely expressed in the cardiovascular system. A study has re- ported that the coding region of Gax gene is quite stable and highly conserved in evolution, so factors other than the coding region may be the main reason for the abnormal expression of the gene. This genetic characteristic is of great significance for maintaining the stable expres-. http://www.ijcem.com. Effect of Gax on the phenotypic transformation of TASMCs. 13266 Int J Clin Exp Med 2019;12(12):13265-13273. sion of Gax in the cardiovascular system . A large number of studies have found that the increasing expression of Gax is significantly cor- related with the decrease of the proliferation and migration of vascular smooth muscle cells (VSMCs), while the increase of the proliferation and migration is closely related to the synthetic transformation of VSMCs phenotype . Ac- cording to a study on the phenotypic transfor- mation of vascular endothelial cells, the expres- sion of Gax in vascular endothelial cells signifi- cantly decreases after the cells are stimulated by mitogens, angiogenic factors, or pro-inflam- matory factors . In view of the regulatory effect of Gax on cells, this study believed that this gene might be one of the key molecules to the phenotypic transformation of TASMCs in TAA. It is urgently needed to make some prog- ress in the pathogenesis of TAA, so as to im- prove the diagnosis and treatment of the dis- ease. However, the mechanism of action of Gax on the phenotypic transformation of TASMCs during the development and progression of the disease has been rarely reported. This study provided a new perspective to understand the phenotypic transformation mechanism of TASMCs in TAA, and deepened the understand- ing of the molecular regulatory mechanism of the development and progression of the dis- ease, which is helpful to explore new targets and to provide new experimental and theoreti- cal basis for the prevention and treatment of TAA.. Materials and methods. Aortic tissue specimens. Sixteen specimens of ascending aortic wall tis- sue were collected from patients with degener- ative ascending thoracic aortic aneurysm (DATAA) who underwent prosthetic vessel re- placement in the Department of Cardiovascu- lar Surgery of Renji Hospital affiliated to Shang- hai Jiaotong University School of Medicine from September 2016 to September 2017. Inclusion criteria: patients diagnosed with fusiform as- cending aortic aneurysm. Exclusion criteria: patients with Takayasu’s arteritis, aortic ath- erosclerosis, autoimmune diseases, heredita- ry aortic diseases such as Marfan syndrome, Loeys-Dietz syndrome and Ehlers-Danlos syn- drome, as well as those with bicuspid aortic valve malformation, familial aortic aneurysm. and syphilis-induced TAA. Further, 16 normal control samples were collected from the proxi- mal anastomosis of bypass grafts in patients with coronary artery disease (CAD) undergoing coronary artery bypass grafting (CABG). In- clusion criteria: patients of the same age as patients with ascending aortic aneurysm. Ex- clusion criteria: no other aortic dilatation dis- eases such as valvular heart disease and aor- tic aneurysm. This study was approved by the Ethics Committee of Renji Hospital affiliated to Shanghai Jiaotong University School of Medi- cine. All enrolled patients and their families signed informed consent forms before surgery.. The samples of ascending aortic aneurysm wall tissue and the normal tissue were rinsed re- peatedly with phosphate buffered saline (PBS) to remove blood clots. Vascular endothelial cells were carefully scraped off and the adven- titia was peeled off with a curved ophthalmic forcep. Next, the tunica media of vascular wall was separated and segmented, and partially frozen in liquid nitrogen for mRNA and protein extraction. And the others were used for prima- ry culture of TASMCs.. Primary culture of TASMCs. In this study, normal human TASMCs and TASMCs from TAA were primarily cultured by digestion and adherence method. The specific steps are as follows: normal ascending aortic wall collected from the proximal anastomosis of bypass grafts during CABG and diseased ascending aortic aneurysm wall removed dur- ing resection surgery were saved in sterile ice Dulbecco’s modified Eagle’s medium (DMEM), then repeatedly rinsed on a clean bench to remove thrombus. The connective tissue of ao- rtic adventitia was removed with an ophthalmic forcep, and the intima, subendothelium of the aorta were bluntly scraped off to obtain the tunica media. The tissue collected was rinsed twice with sterile PBS buffer to remove residual vascular endothelial cells and fibroblasts. The aortic media was digested with 0.25% trypsin solution for 20 min. 10% fetal bovine serum (FBS) was added to terminate the digestion and then discarded. Afterwards, the tissue was placed in a culture dish and moistened with DMEM containing 20% FBS. After cut into nee- dle-tip size with an aseptic ophthalmic curved scissors, the tissue blocks were evenly distrib-. Effect of Gax on the phenotypic transformation of TASMCs. 13267 Int J Clin Exp Med 2019;12(12):13265-13273. uted at the bottom of the culture dish at a spac- ing of 0.5 cm. When the blocks firmly adhered to the bottom of the culture dish, DMEM con- taining 20% FBS was slowly added along the wall. Avoid removing the blocks from the bot- tom when adding the liquid and ensure that they were completely immersed in the culture dish. Subsequently, the blocks were placed in a 37°C, 5% CO2 incubator for 3 days. After cells swam out from the edge of the tissue block, the half or 2/3 of medium was exchanged every 3 days.. Immunofluorescence staining. Primary cultured normal TASMCs and TASMCs from TAA were seeded into a six-well culture dish. When reaching 70% confluence, the cells were rinsed twice with PBS for 5 min each time, fixed with 4% paraformaldehyde for 30 min and washed 3 times with PBS buffer for 2 min each time. Then 0.1% Triton-X-100 was added, the cells were incubated at 4°C for 10 min to increase membrane permeability and washed twice with PBS for 3 min each time. 10% nor- mal sheep serum blocked the cells for 30 min and discarded. The cells were incubated with mouse anti-human α-actin antibody (1:500) overnight at 4°C, then washed 3 times with PBS for 5 min each time. Goat anti-mouse IgG (1:1,000) was added and incubated at 37°C for 1 h in the dark, washed 3 times with PBS for 5 min each time. The nucleus was stained with DAPI (1:1,000) for 3 min, washed twice with PBS for 5 min each time, then observed and photographed under a fluorescence microsco- pe.. Western blot. Cells or crushed aortic wall tissue samples were collected and mixed with ice-precooled protein lysate, centrifuged at 12,000 rpm for 5 min at 4°C, and then the supernatant was extracted. The protein content was determined by bovine serum albumin (BSA). After preparing separating gel and stacking gel, samples were electophoresised by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) at 60 V. When bromophenol blue reached the junction of concentrated gel and stacking gel, the voltage was changed to 120 V, and the electrophoresis was terminated when bromo- phenol blue came out of the gel. The proteins were transferred to a polyvinylidene difluoride. (PVDF) membrane at 60 V for 2 h. The mem- brane was rinsed with TBST buffer and the immune reaction was carried out. The chemical developer was dropped onto the membrane. The protein bands on the membrane were developed in a light-exposure apparatus, and the optical density was analyzed by gel image processing system.. Real-time PCR. Cells or crushed aortic wall tissue samples were collected, and total RNAs were extracted with Trizol lysate. Primer Premier 5.0 software was used to design and synthesize primers: Gax: 5’-CCAGACTGAGGCGATACGAG-3’ (forwa- rd); 5’-CTGTTTGCTGGAGGGTGGC-3’ (reverse). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 5’-GGGTGTGAACCATGAGAA GTATG-3’ (forward), 5’-GATGGCAT GGACTGTGGTCAT-3’ (re- verse). A 20 μL reverse transcription reaction system was prepared according to the instruc- tions of the reaction kit: 4 μL of 5 × Buffer, 4 μL of MgCl2, 2 μL of dNTP, 1.0 μL of reverse tran- scriptase, 0.5 μL of RNase, 1 μg of RNA, 1 μL of Oligo-dT, made up to the final volume with ddH2O. Reverse transcription reaction condi- tions: 42°C for 60 min, 99°C for 5 min. cDNA was stored at -80°C for later use. A 15 μL qRT- PCR reaction system was prepared: 0.2 μL of each upstream and downstream primer, 1 μL of cDNA, 7.5 μL of 5 × SYBR Premix Ex TaqTM, made up to the final volume with ddH2O. The reaction conditions are as follows: pre-denatur- ation at 95°C for 3 min, denaturation at 95°C for 25 s, annealing at 58°C for 45 s, extension at 72°C for 2 min, for a total of 35 cycles. The target gene was quantified using ΔΔCt method, and the relative expression was calculated by 2-ΔΔCt.. Transfection of TASMCs. TASMCs in logarithmic growth phase were inoc- ulated into a culture dish, and the DMEM con- taining 10% FBS was replaced with DMEM con- taining 1% FBS when cells reached 70-80% confluence. Adenoviral transduction of the Ad- Gax transfection regent was added and the cells were placed in a 37°C, 5% CO2 incubator for 4 h. In order to make the regent transfect cells evenly, the culture dish was shaken once every 15 min. The TASMCs were washed with PBS and placed in a 37°C, 5% CO2 incubator for subsequent experimental detection.. Effect of Gax on the phenotypic transformation of TASMCs. 13268 Int J Clin Exp Med 2019;12(12):13265-13273. Proliferation of TASMCs. In this study, CCK-8 kit was employed to detect the proliferation of TASMCs. The specific steps are as follows: the TASMCs were transfected with Ad-Gax over-expression vector. The TAS- MCs in logarithmic phase were digested with 0.25% trypsin and prepared into single cell sus- pension, which was seeded into a 96-well cul- ture plate with 2 × 103 cells per well and a final volume of 200 µL per well. The cells were cul- tured for 24 h, 48 h, 72 h, and 96 h after they were completely adherent to the wall. At differ- ent time points, 20 µL CCK-8 solution was added to each well, and the cells were continu- ously incubated at 37°C for 2 h. The optical density (OD) value in each well was read by a microplate reader at 450 nm wavelength to determine the cell proliferation.. Migration of TASMCs. In this study, Transwell was employed to detect the cell migration of TASMCs. The specific steps are as follows: the TASMCs were transfected with Ad-Gax over-expression vectors. When the cells reached 80% confluence, serum-free DMEM culture solution was used to synchro- nize TASMCs for 24 h. The cells were digested with 0.25% trypsin and prepared into single cell suspension with a density of about 3 × 105 cells/mL. A total of 100 μL of cell suspension. was added to the upper chamber of Transwell migration system (24-well plate), 600 μL of DMEM containing 10% FBS was added to the lower chamber, and the cells were cultured in a 37°C, 5% CO2 incubator for 6 h. TASMCs with- out migration in the upper chamber were care- fully wiped off with cotton swabs, and those reaching the bottom of the cup were stained with 0.1% crystal violet. Under an inverted mi- croscope, 10 fields (100 ×) were randomly selected for observation and photographing, and the number of migrated cells was calcula- ted.. Statistical analysis. SPSS 20.0 was used to analyze all the data. The measurement data were expressed by means ± standard deviation. The comparison between the two groups was conducted by independent sample t test, while the compari- son among more than three groups (including three groups) was conducted by one-way analy- sis of variance. The counting data were ex- pressed by percentage or rate. The comparison between the groups was conducted by chi- square test. A value of P<0.05 was considered statistically significant.. Results. Difference of Gax expression in tunica media of thoracic aortic aneurysm wall and normal aortic wall. Real-time PCR results showed that compared with the tunica media of normal aortic wall, the expression of Gax mRNA reduced significantly in tunica media of thoracic aortic aneurysm wall (P<0.05). See Figure 1.. Western blot results showed that the expres- sion of Gax protein in the tunica media of tho- racic aortic aneurysm wall was significantly lower than that in the tunica media of normal thoracic aortic wall (P<0.05). See Figure 2.. Primary culture of TASMCs. After 1 week of culturing the aortic media blocks by digestion and adherence method, cells crawled out of the edge of the blocks. Normal TASMCs, collected from the proximal anastomosis of bypass grafts in patients with CAD undergoing CABG, were fusiform, with cell polarity and vaguely visible myofilaments. Whe-. Figure 1. Real-time PCR detection of Gax expression in tunica media of aortic wall. Compared with normal group, *P<0.05. Gax, growth arrest-specific homeo- box; DATAA, degenerative ascending thoracic aortic aneurysm.. Effect of Gax on the phenotypic transformation of TASMCs. 13269 Int J Clin Exp Med 2019;12(12):13265-13273. reas TASMCs in surgically resected diseased tissue of patients with ascending aortic aneu- rysm were irregular in shape and weak in cell polarity. See Figure 3.. The CCk8 method showed that at culture time points of 48 h, 72 h and 96 h, the OD value of TASMCs with over-expressed Gax was signifi- cantly lower than that in the control group and. Figure 2. Western blot detection of Gax protein expression in tunica media of aortic wall. A. Protein bands. B. Relative expression of Gax protein. Com- pared with normal group, *P<0.05. Gax, growth arrest-specific homeobox; DATAA, degenerative ascending thoracic aortic aneurysm.. Figure 3. VSMCs culture by digestion and adherence method. A. VSMCs in normal thoracic aortic; B. VSMCs in thoracic aortic aneurysm. VSMCs, vascular smooth muscle cells.. Figure 4. Detection of α-actin expression in VSMCs by immunofluorescence staining. A. VSMCs in normal thoracic aortic; B. VSMCs in thoracic aortic aneurysm. VSMCs, vascular smooth muscle cells.. Immunofluorescence staining of α-actin. Immunofluorescence staining results of α-actin showed that the α-actin protein was signifi- cantly expressed in cultured cells (red fluorescence), indi- cating that they were aortic smooth muscle cells. From Figure 4, it can be seen that the expression of α-actin in normal TASMCs was signifi- cantly higher than that in TASMCs from thoracic aortic aneurysm tissue, indicating that the TASMCs from TAA were transformed from con- tractile phenotype to synthe- tic phenotype.. Expression of Gax in TASMCs. The expression of Gax in TA- SMCs was mainly concentr- ated in nucleus (red fluores- cence). Compared with nor- mal TASMCs, those from tho- racic aortic aneurysm tissue showed significantly lower Gax expression. See Figure 5.. Effects of Ad-Gax transfection on expressions of Calpoin and SM-MHC 11 proteins in TASMCs. Western blot showed that compared with the control group and Ad-EGFP transfect- ed group, Ad-Gax transfecti- on over-expressed the Gax in TASMCs, which up-regulated the expressions of Calpoin and SM-MHC 11 proteins. See Figure 6.. Effects of Gax over-expression on proliferation of TASMCs. Effect of Gax on the phenotypic transformation of TASMCs. 13270 Int J Clin Exp Med 2019;12(12):13265-13273. Ad-EGFP transfected group (both P<0.05), whi- le the difference in TASMC proliferation be- tween the control group and Ad-EGFP transfect-. control group and Ad-EGFP transfection group. See Figure 8.. Discussion. TAA is one of the serious cardiovascular diseas- es with hidden onset and poor prognosis. Researches on its etiology and pathogenesis may provide guidance for the discovery of high- risk groups, early prevention and treatment. Recent studies have shown that the occurrence and development of TAA are closely related to atherosclerosis, hypertension, aortitis, and genetic diseases [11, 12], but the exact patho- genesis still remains unclear. Researches on the pathogenesis of TAA from clinical, patho- logical and molecular biology indicate that it is caused by a series of different pathological pro- cesses, among which the phenotypic transfor- mation of TASMCs is one of the precursors of TAA [13, 14]. Normal TASMCs express markers of contractile phenotype in the tunica media. Moreover, they can make characteristic reac- tive contraction for vascular wall stretch caused by pulsatile flow, which is regulated by periodic contraction of thick and fine filaments [15, 16]. The thick filament is composed of β-myosin and the thin filament is composed of α-actin. Studies have shown that the decrease of vaso- constriction caused by synthetic transforma- tion of VSMCs plays an important role in the pathological dilatation of elastic artery [17, 18]. α-actin is one of the commonly recognized markers of contractile phenotype of VSMCs [19, 20]. The results of this study found that the expression of α-actin protein in TASMCs from TAA was significantly lower than that in normal TASMCs, which further indicated that pheno-. Figure 5. Immunofluorescence staining of Gax in VSMCs. A. VSMCs in nor- mal thoracic aortic; B. VSMCs in thoracic aortic aneurysm. VSMCs, vascular smooth muscle cells; Gax, growth arrest-specific homeobox.. Figure 6. Effect of Gax overexpression on the expres- sion of contractile VSMCs marker proteins Calpoin and SM-MHC 11. VSMCs, vascular smooth muscle cells; Gax, growth arrest-specific homeobox.. Figure 7. Effect of Gax overexpression on VSMCs pro- liferation at different culture time points. Compared with control group and Ad-EGFP transfection group, *P<0.05. VSMCs, vascular smooth muscle cells; Gax, growth arrest-specific homeobox.. ed group was not statistically significant. See Figure 7.. Effects of Gax over-expression on migration of TASMCs. The Transwell assay showed that the number of migrated TASMCs with over-expressed Gax was significantly less than that in the control group and Ad-EGFP transfected group (both P<0.05), while there was no statistically significant dif- ference in the number of mi- grated TASMCs between the. Effect of Gax on the phenotypic transformation of TASMCs. 13271 Int J Clin Exp Med 2019;12(12):13265-13273. typic transformation of TASMCs was closely related to the occurrence of TAA. In addition, a study revealed that SM22α expression was down-regulated and MMP-2 and MMP-9 expres- sions were up-regulated in TAA, which also sug- gested that phenotypic transformation of TA- SMCs was one of the early events of TAA and participated in its pathological process .. Embryonic transcription factor Gax is mainly expressed in skeletal, smooth and cardiac muscles and plays a role in the growth and development of various muscle tissues . While adult Gax is mainly expressed in heart, kidney, lung and other organs and tissues rich in blood vessels. In recent years, Gax is consid- ered as one of the nuclear transcription factors regulating the biological behavior of VSMCs and plays an important role in the phenotypic transformation of VSMCs. Previous studies have reported that phenotypic transformation of TASMCs is the key to the occurrence and pro- gression of TAA, but the role of Gax in TAA has not been explored. Therefore, this study aimed to confirm the relationship between the two and to explore the possible molecular mecha- nism.. Gax gene was isolated from cDNA library of rat aorta by Gorski et al. in 1993 . The human Gax gene is located at 7P21 and encodes 302 amino acids with a cDNA length of 941 bp. Compared with rat Gax gene, the homology of human Gax gene was 83% and 98%, at nucleo- tide and amino acid levels, respectively . Our study showed that over-expression of inhib- itory transcription factor Gax maintained the contractile phenotype of TASMCs, and leaded to a decrease in cell proliferation and migra-. tion, indicating that Gax may be one of the key factors regulating contractile phenotype of TASMCs. A study demonstrated a significant down-regulation of Gax expression in VSMCs of vascular injury models . Another study found that Gax mRNA expression in VSMCs was significantly down-regulated under stimulation of mitogens such as platelet-derived growth factor-BB (PDGF-BB), which could promote cell proliferation and migration . Therefore, the Gax expression was down-regulated in VSMCs stimulated by pathological conditions, and pro- moted cell proliferation and migration by regu- lating expression of downstream genes, there- by playing a role in pathological process of inti- mal hyperplasia after vascular injury. Our study also found that Gax was induced to be down- regulated in TAA, and the over-expression of Gax could significantly change the phenotypic transformation of TASMCs.. To sum up, the Gax expression in the tunica media of thoracic aortic aneurysm wall is sig- nificantly lower than that in normal aortic tis- sue. The Gax expression in TASMCs from TAA is significantly lower than that in normal TASMCs. Over-expression of Gax in TASMCs can signifi- cantly up-regulate the expression of contractile marker of TASMCs, accompanied by reduction of TASMC proliferation and migration, which indicates that Gax is of great significance in the phenotypic transformation of TASMCs, and its down-regulation may play a role in the occur- rence and development of TAA by promoting the transformation of TASMCs to synthetic phe- notype. However, the specific molecular mech- anism by which Gax regulates TASMC pheno- typic transformation needs to be further stu- died.. Acknowledgements. This work was supported by Grants from the National Natural Science Foundation of China (8150036).. Disclosure of conflict of interest. None.. Address correspondence to: Song Xue, Department of Cardiovascular Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, No. 160 Pujian Road, Shanghai 200127, China. Tel: +86- 021-68383704; Fax: +86-021-68383704; E-mail: email@example.com. Figure 8. Effect of Gax overexpression on VSMCs migration. Compared with control group and Ad- EGFP transfection group, *P<0.05. VSMCs, vascular smooth muscle cells; Gax, growth arrest-specific ho- meobox.. mailto:firstname.lastname@example.org. Effect of Gax on the phenotypic transformation of TASMCs. 13272 Int J Clin Exp Med 2019;12(12):13265-13273. References.  Boczar KE, Cheung K, Boodhwani M, Beauchesne L, Dennie C, Nagpal S, Chan K and Coutinho T. Sex differences in thoracic aortic aneurysm growth: role of aortic stiff- ness. Hypertension 2019; 73: 190-196..  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