Irisin/PPARá Mediates the Proliferation and Apoptosis of Ascular Smooth Muscle Cells through Activation of the HO-1

doi:10.21203/rs.3.rs-1870612/v1

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Irisin/PPARá Mediates the Proliferation and Apoptosis of Ascular Smooth Muscle Cells through Activation of the HO-1

https://doi.org/10.21203/rs.3.rs-1870612/v1

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Objective: This study aimed to investigate the function and mechanism that Irisin/ PPARαsignaling pathway mediates the balance between proliferation and apoptosis in vascular smooth muscle cells (VSMCs).

Methods: VSMCs were transfected with FNDC5 overexpression and silencing lentiviral vectors to increase or inhibit Irisin production; the cells were randomly divided into 7 groups:the control group (control), overexpressing empty lentiviral vector group (MOCK1), FNDC5 overexpressing group (FNDC5), overexpressing empty vector with PPARα inhibitor group (MOCK1 + GW6471), FNDC5 overexpressing with PPARα inhibitor group (FNDC5 + GW6471), silencing empty lentiviral vector group (MOCK2) and FNDC5 silencing group (sh- FNDC5).The expression levels of FNDC5, PPARα, HO-1, Bax, and Bcl-2 in each group were detected by Real-time PCR and Western blot, respectively. The Proliferation and Apoptosis of VSMCs were conducted by using flow cytometry. The gell proliferation rate was detected through CCK8 assay.

Results: Compared with the control group or MOCK1 group, the expressions of FNDC5, PPARα, HO-1 and Bax，and the Bax/Bcl-2 ratio，increased significantly in FNDC5 and FNDC5+GW6471 group(P < 0.05) , while the expression of Bcl-2 decreased significantly in MOCK+GW6471 group (P < 0.05).

Conclusions:The HO-1 induced by Irisin/PPARα,inhibited the proliferation of VSMCs through repressing DNA synthesis ,and regulated expressions of the important functional molecules in the cell apoptosis pathway to promote the cell death，therefore, we drew a conclusion that Irisin/PPARα mediates the the balance between proliferation and apoptosis of ascular smooth muscle cells through activing the HO-1.

Irisin

PPARα

HO-1

VSMCs

Proliferation

Apoptosis

Atherosclerosis is the main pathological basis of cardiovascular diseases, which is one of the primary cause of human death. Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a key event during the atherosclerosis development, and a prominent pathological feature of atherosclerosis and restenosis. The imbalance between proliferation and apoptosis of VSMCs plays a crucial role in the occurrence of angioproliferative diseases such as atherosclerosis and restenosis[1,2].In early atherosclerosis,the number of VSMCs increases and migrates from the media to the intima due to the low level of cell proliferation and high level of apoptosis, causing wall thickening, artery stenosis and fibrous cap formation[3-8].In late atherosclerosis, excessive VSMCs apoptosis induce thinning,destabilization,and rupture of fibrous cap of plaques, triggering cardiovascular and cerebrovascular events.Meanwhile,that apoptotic VSMCs cannot be removed in time , which caused by the reduced phagocytic ability of macrophages in advanced atherosclerosis, leading to secondary inflammation and necrosis to aggravate atherosclerosis[9,10].Therefore, maintaining the imbalance between proliferation and apoptosis of VSMCs, have very important clinical significance for the prevention and treatment of angioproliferative diseases such as atherosclerosis and restenosis[11]。

Irisin, a 112 amino acid polypeptide hormone produced from proteolytic processing and glycosylation of fibronectin type IIIdomain containing 5 (FNDC5),in modulation of metabolism and protection against obesity has anti-inflammatory and oxidative effects, can inhibit atherosclerosis through regulating glucose and lipid metabolism to improve obesity and insulin resistance[12] and alleviating oxidative stress and inflammation. We have confirmed that serum irisin levels are significantly reduced in patients with coronary heart disease[13,14]，and Irisin can suppress the development of atherosclerosis through anti-inflammatory and oxidative stress[13,14]. The research has shown that Irisin inhibit VSMCs proliferation induced by platelet-derived growth factor-BB[15].Preliminary exploration in this study revealed that transfection of VSMCs with overexpressing FNDC5 inhibited their proliferation and migration,the opposite result by silencing FNDC5 [ 王向 ].Peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated nuclear transcription factor in the nuclear receptor superfamily. PPARα,a downstream gene of FNDC5,mediates uncoupling protein-1 (UCP1) to transform white adipose tissue into brown adipose tissue, called white fat browning.FNDC5 increased UCP1 expression and enhanced the browning effectby upregulating PPARα expression, but it could be significantly attenuated by antagonists of PPARα, indicating that the regulatory effect of FNDC5 on fat metabolism is at least partially achieved by PPARα[16].PPARα expresses in atherosclerotic plaques regulates cell glucose and lipid metabolism, with the function of anti-inflammatory and cardiovascular protective effects[16].The increase of expression of PPARαinhibits VSMCs proliferation and promotes their apoptosis[18].Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in heme catabolism and can decompose heme into CO, Fe3 +, and biliverdin. Both HO-1 and CO inhibit VSMCs proliferation, antioxidant, inflammatory and atherosclerotic.The above role of HO-1/CO system was also confirmed by our previous study[19-21].HO-1 is a direct target gene of PPARα, which can induce HO-1 expression ,and inhibit inflammatory response and VSMCs proliferation[22]. therefore, the effects of anti-inflammation,against aoxidation,inhibition of VSMCs proliferation and promotion of their apoptosis lay the foundation for the anti-atherogenesis of irisin, PPARα and HO-1.

However, the molecular mechanism by which irisin inhibits the proliferation and anti- atherosclerosis of VSMCs is still unclear.In addition, there has not been reported that the irisin/PPARαpathway can mediate HO-1 to regulate VSMCs proliferation and apoptosis. The aim of this study is to explore the function and mechanism of irisin/PPARα/HO-1 signaling pathway in regulating the balance between proliferation and apoptosis of VSMCs on the basis of previous studies, providing a theoretical and experimental basis for the clinical prevention and treatment of angioproliferative diseases such as atherosclerosis and restenosis.

1.1 Main reagents

Mouse aortic VSMCs were purchased from Procell Life Science&Technology Co.Ltd.pLVX-mCMV-ZsGreen-IRES-Puro lentiviral overexpression vector, pLKD-CMV- GampPR-U6-shRNA vector and 293T cell line were purchased from Wuhan Viraltherapy Technologies Co.Ltd. Reverse transcription kit, SYBR Green Master Mix kit and purchased from Nanjing Vazyme Biotech Co.,Ltd. Cell culture medium DMEM/F12, fetal bovine serum (FBS), penicillin-streptomycin mixture purchased from Gibco, USA.KFluor647-EdU cell proliferation detection kit, cell cycle and apoptosis detection kit were purchased from Nanjing KeyGen Biotech.Inc.cell counting kit (CCK-8) was purchased from BIOSHARP.PPARα-specific inhibitor GW6471 was purchased from Sigma, USA; β-actin antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd.; FNDC5 antibody was purchased from BEIJING BIOSYNTHESIS BIOTECHNOLOGY CO., LTD.PPARα, HO-1, and Bax antibodies were purchased from Proteintech Group, Inc.Bcl-2 antibody was purchased from abcam, USA.Primer synthesis and sequencing were completed by Beijing Optimus Biotechnology Co., Ltd.

1.2 Construction of FNDC5 gene overexpression and silencing lentiviral vector[23]

The FNDC5 cDNA clone was synthesized by PCR and recombined into the pLVX-mCMV- ZsGreen-IRES-Puro overexpression vector, and then transformed into 293T competent cells to obtain the recombinant lentivirus using a four-plasmid packaging system. The FNDC5 shDNA fragment was recombined and cloned into the pLKD-CMV-GampPR-U6-sh RNA vector, and the recombinant lentivirus was also obtained by using a four-plasmid packaging system.

1.3 Culture and treatment of VSMCs

VSMCs were resuspended in DMEM/F12 complete medium containing 15% FBS, 1% penicillin- streptomycin.The cells were gently resuspended and transferred into 25cm² cell culture flasks, cultured at 37℃ in a 5% CO2 cell incubator, and subcultured at a cell number of 1 × 108.The cells were divided into seven groups and treated as followed:Control group added ox-LDL,MOCK1 group added ox-LDL and transfected with overexpression lentiviral empty vector, FNDC5 group added ox-LDL and transfected with FNDC5 gene overexpression vector, MOCK1 + GW6471 group added PPARα inhibitor GW6471 on the basis of MOCK1 group, FNDC5 + GW6471 added PPARα inhibitor GW6471 on the basis of FNDC5 group, MOCK2 group added ox-LDL and transfected with shRNA lentiviral empty vector, sh-FNDC5 added ox-LDL and transfected with sh-FNDC5 lentivirus.

Relevant treatments were performed, fluorescence labeling was observed under a fluorescence microscope, and stable VSMCs strains were selected with puromycin.

1.4 Real-time PCR was used to detect FNDC5, PPARα, HO-1, Bax, Bcl-2, and caspase-3 mRNA expression levels in VSMCs

Total RNA was extracted by Trizol method, cDNA was synthesized by reverse transcription, and then real-time quantitative polymerase chain reaction (qRT-PCR) was performed using SYBR Green Master Mix kit, and non-specific amplification was excluded by melting curve and amplification curve analysis. Reaction conditions: pre-denaturation at 95℃ for 10 min, denaturation at 95℃ for 30 seconds, annealing at 60 ° C for 30 seconds, and 40 cycles. The experiment was repeated three times for each sample.β-actin was used as an internal reference gene, and the relative expression level of the target gene was calculated with the 2-△△Ct method (△Ct= mean Ct value of target gene in the experimental group - mean Ct value of target gene in the control group - mean Ct value of internal reference gene).Upstream primer sequence of internal reference gene β-actin：5’- CACGATGGAGGGGCCGGACTCATC-3’，downstream primer sequence：5’- TAAAGACCTCTATGCCAACACAGT-3’ 。 Upstream primer sequence of target gene FNDC5:5 ‘ - TGAAGGAGATGGGGAGGAAC-3 ， downstream primer sequence：5 ‘ -TGGTTTCTGATGCGCTATTG-3 ’；PPARα Upstream Primer Sequence:5 ‘ - AGACAAAGAGGCAGAGGTCC-3 ’，downstream primer sequence：5 ‘ - CGATCAGCATCCCGTCTTTG-3 ’ ； HO-1 Upstream Primer Sequence:5 ‘ - CCTCACTGGCAGGAAATCA-3 ’ ， downstream primer sequence：5 ‘ - TCGGGAAGGTAAAAAAAGC-3 ’；Bax Upstream Primer Sequence:5 ‘ - TTTTGCTACAGGGTTTCATCCA-3 ’，downstream primer sequence：5 ‘ - GTGTCCACGTCAGCAATCATC-3 ’；Bcl-2 upstream primer sequence:5 ’TCATGAAGACAGGGGCCTTT-3 ’，downstream primer sequence:5 ’

-GTCCACGTCAGCAATCATCC-3’。

1.5 FNDC5, PPARα, HO-1, Bax, Bcl-2, and caspase-3 protein expression levels in VSMCs were measured by Western blot

RIPA lysate was added into the each group cells on ice,and the supernatant was obtained after centrifugation for protein quantification by using the BCA method. Equal amounts of protein were loaded and subjected to SDS-PAGE electrophoresis, then was constantly circulated to transfer to a PVDF membrane and blocked for 1 h at room temperature. After blocking, rabbit polyclonal antibodies FNDC5 (1:1000), PPARα (1:600), HO-1 (1:3000), Bax (1:5000), Bcl-2 (1:600), and β-actin primary antibodies (1:200) were added, respectively, and incubated overnight at 4℃, and TBST membranes were washed three times for 15 min each, followed by goat anti-rabbit IgG (1:5000) for 1 h at room temperature.Protein expression was detected by ECL, β-actin was used as an internal reference, and the ratio of target protein to its gray level indicated the relative expression of target protein.Band gray value analysis was performed by using Gel-Pro analyzer4 software.

1.6 Proliferative activity of VSMCs detected by CCK-8

Mouse aortic smooth muscle cells in logarithmic growth phase and in good growth state were inoculated into a 96-well plate at 5 × 103 cells/well.200μl of culture medium was added to each well, cultured in an incubator containing 5% CO₂ at 37℃.CCK-8 solution was added at 72 h after transfection, and OD450 was measured by a microplate reader 4 h later.

1.7 The proliferation rate of VSMCs measured by flow cytometry

VSMCs were seeded in 6-well plates, incubated with ox-LDL for 72 h, and then cultured with 50 μM EdU for 2 h[24]. The cells were resuspended with 1 ml of PBS containing 4% paraformaldehyde, mixed well, and incubated in the dark at room temperature for 15 min. The cells were resuspended with 500 μl of PBS containing 0.3% Triton X-100, mixed well, and incubated for 20 min. 1mL of Click-iT reaction mixture (prepared according to the instructions of the kit) was added to each tube, mixed well, and the reaction mixture was incubated in the dark at room temperature for 30 min. The cells were detected by flow cytometry. The experiment was independently repeated three times.

1.8 Detection of Proliferation Cycle of VSMCs by Flow Cytometry

Cells were divided into groups, and VSMCs from each group were collected 72 hours after lentiviral transfection, rinsed with PBS, and then stained with 50 mg/ml propidium iodide for 30 min at 4 ° C and detected on a flow cytometer. The experiment was repeated three times.

1.9 Detection of VSMCs apoptosis by flow cytometry

72 hours after lentiviral transfection,500 μl Binding Buffer was added to resuspend the VSMCs (3 × 105/ml) collected from each group.5 μl AnnexinV-APC was added and mixed well, and then 5 μl 7-AAD was added and mixed well, reaced at room temperature in the dark for 5-15 min, and detected on the flow cytometer. The experiment was independently repeated three times.

1.10 Statistical processing

The data were analyzed by SPSS 20.0 and GraphPad Prism7 statistical software. Measurement data were expressed as mean ± standard deviation (x ± s). T-test was used for comparison between two groups. One-way analysis of variance was used for comparison between multiple groups. SNK-q test was used for pairwise comparison. P < 0.05 was considered statistically significant.

2.1 Lentivirus-transfected VSMCs and selection of stable cell lines

Seventy-two hours after lentivirus (MOI = 30) transfection of VSMCs, most of the cells were seen to emit green fluorescence, with transfection efficiency(78.30 ± 6.10)% （Fig.1）. Puromycin at 1μg/mL was added for selection, and the cells had no obvious morphological changes, grew well, and green fluorescence continued to appear stably, resulting in a VSMCs strain with stable FNDC5 expression.

2.2 The influence of overexpression of FNDC5, silencing FNDC5 and down regulating PPARαon the basis of overexpression of FNDC5 on FNDC5, PPARα, and HO-1 mRNA and protein expression in VSMCs

Compared with the control group, there was no statistically significant difference in the mRNA and protein expression levels of FNDC5, PPARα, and HO-1 in the MOCK1 group (all P>0.05).Compared with control group and MOCK1 group, the expression of FNDC5, PPARα, HO-1 mRNA and protein in FNDC5 group and FNDC5 + GW6471 group was significantly increased (all P＜0.05).Compared with control group and MOCK1 group,the expression of PPARα and HO-1 mRNA and protein was significantly decreased in the MOCK1 + GW6471 group (all P＜0.05), and the expression of FNDC5 mRNA and protein was not significantly changed in the MOCK1 + GW6471 group (all P＞ 0.05).Compared with MOCK1 + GW6471 group, the expressions of FNDC5, PPARα, HO-1 mRNA and protein in FNDC5 group and FNDC5 + GW6471 group were significantly higher (all P

＜0.05).Compared with FNDC5 group, the expression of PPARα and HO-1 mRNA and protein in FNDC5 + GW6471 group was significantly decreased (all P < 0.05), but the expression of FNDC5 mRNA and protein was not significantly changed (all P > 0.05).Compared with the control group and MOCK2 group, the expressions of FNDC5, PPARα, HO-1 mRNA and protein in the sh-FNDC5 group were significantly decreased (all P＜ 0.05), and the expressions of FNDC5 mRNA and protein were not significantly changed (all P＞0.05). It is shown in Figures 2 and 3.

2.3 The influence of overexpression, silencing FNDC5 and down regulating PPARαon the basis of overexpression of FNDC5 on Bax , Bcl-2 mRNA and protein expression in VSMCs(供参考)

Compared with the control group, there was no significant difference in the expression levels of Bax, Bcl-2 mRNA and protein, and Bax/Bcl-2 ratio in the MOCK group (all P＞0.05).Compared with the control group and MOCK group, the expression levels of Bax mRNA and protein, Bax/Bcl-2 ratio were significantly increased, and the expression levels of Bcl-2 mRNA and protein were significantly decreased in the FNDC5 group and F NDC5 + GW6471 group (all P < 0.05)；in addition, the expression levels of Bax mRNA and protein, Bax/Bcl-2 ratio were significantly decreased, and the expression levels of Bcl-2 mRNA and protein were increased in the MOCK + GW6471 group (all P < 0.05).Compared with MOCK + GW6471 group, the expression levels of Bax mRNA and protein, Bax/Bcl-2 ratio were significantly increased, and the expression levels of Bcl-2 mRNA and protein were decreased in FNDC5 group and FNDC5 + GW6471 group (all P＜0.05).Compared with FNDC5 group, the expression levels of Bax mRNA and protein, and Bax/Bcl-2 ratio were significantly decreased, and the expression levels of Bcl-2 mRNA and protein were increased in FNDC5 + GW6471 group (all P < 0.05).Compared with control group and MOCK2 group, the expression levels of Bax mRNA and protein, and Bax/Bcl-2 ratio were significantly decreased, and the expression levels of Bcl-2 mRNA and protein were significantly increased in sh-FNDC5 group (all P < 0.05).It is shown in Figures 4 and 5.

2.4 Overexpression of FNDC5, silencing FNDC5 and down- regulation of PPARα expression on the basis of overexpression of FNDC5 on the VSMCs proliferation activity

Compared with the control group, there was no statistically significant difference in the proliferative activity of VSMCs in the MOCK group (P > 0.05).Compared with control group and MOCK group, the proliferation activity of VSMCs in FNDC5 group and FNDC5 + GW6471 group as significantly decreased (P < 0.05), and the proliferation activity of VSMCs in MOCK + GW6471 group was significantly increased (P > 0.05).Compared with MOCK + GW6471 group, the proliferation activity of VSMCs in FNDC5 group and FNDC5 + GW6471 group was significantly decreased (P < 0.05).Compared with the FNDC5 group, the proliferation activity of VSMCs was significantly increased in the FNDC5 + GW6471 group (P < 0.05).Compared with control group and MOCK2 group, the proliferation activity of VSMCs in sh-FNDC5 group was significantly increased (P > 0.05).It is shown in table 1.

Table 1

Comparison of proliferation activity of VSMCs in each group（`x±s）

Compared with the control group,aP＜0.05，n=3; compared with the MOCK1 group,bP ＜0.05,n=3；compared with the MOCK + GW6471 group,cP＜0.05，n=3;compared with the FNDC5 group,dP＜0.05, n=3；compared with the MOCK2 group,eP＜0.05,n=3

2.5 Overexpression of FNDC5, silencing FNDC5 and down- regulation of PPARα expression on the basis of overexpression of FNDC5 on the proliferation rate of VSMCs

Compared with the control group, there was no statistically significant difference in the proliferation rate of VSMCs in the MOCK group (P > 0.05).Compared with control group and MOCK group, the proliferation rate of VSMCs in FNDC5 group and FNDC5 + GW6471 group was significantly decreased (P < 0.05), and the proliferation rate of VSMCs in MOCK + GW6471 group was significantly increased (P < 0.05).Compared with MOCK + GW6471 group, the proliferation rate of VSMCs in FNDC5 group and FNDC5 + GW6471 group significantly decreased (P＜0.05).Compared with FNDC5 group, the proliferation rate of VSMCs in FNDC5 + GW6471 group was significantly increased (P < 0.05).Compared with the control and MOCK2 groups, the proliferation rate of VSMCs in the sh-FNDC5 group significantly increased (P＜0.05).Compared with the control and MOCK2 groups, the proliferation rate of VSMCs in the sh-FNDC5 group significantly increased (P < 0.05).It is shown in Figures 6.

2.6 Overexpression of FNDC5, silencing FNDC5 and down-regulation of PPARα expression on the basis of overexpression of FNDC5 on the apoptosis rate of VSMCs

Compared with the control group, there was no statistically significant difference in the apoptosis rate of VSMCs in the MOCK group (P > 0.05).Compared with control group and MOCK group, the apoptosis rate of VSMCs in FNDC5 group and FNDC5 + GW6471 group was significantly increased (P < 0.05), and the apoptosis rate of VSMCs in MOCK + GW6471 group was decreased (P < 0.05).

Compared with MOCK + GW6471 group, the apoptosis rate of VSMCs in FNDC5 group and FNDC5 + GW6471 group increased (P < 0.05).Compared with FNDC5 group, the apoptosis rate of FNDC5 + GW6471 group was decreased (P < 0.05).Compared with the control and MOCK2 groups, the apoptosis rate of VSMCs in the sh-FNDC5 group was significantly lower (P < 0.05).It is shown in Figures 7.

Atherosclerosis, endothelial denudation, and restenosis after angioplasty have been outstanding clinical problems.The imbalance between VSMCs proliferation and apoptosis is an important cause of angioproliferative diseases such as atherosclerosis and restenosis. Reducing VSMCs proliferation and increasing their apoptosis can reduce ox-LDL-induced vascular injury[25],and targeted promotion of apoptosis in VSMCs may be an effective way to reduce the occurrence of atherosclerosis and restenosis[26].Bcl-2 is a key factor in the apoptosis signaling pathway, and up-regulation of Bax protein, a homologous gene of Bcl-2, can promote apoptosis, which eventually leads to apoptosis after step-by-step activation of apoptotic proteases caspase-3 and caspase-9.Caspase-3 is considered as a marker for the development of apoptosis. The key factors of apoptosis, Bax and Bcl-2, maintain the balance of apoptosis through forward and reverse regulation, respectively[27].It is of great significance to actively explore new targets that can effectively regulate the balance of proliferation and apoptosis of VSMCs for the prevention and treatment of atherosclerosis and restenosis.Irisin improves obesity and insulin resistance by regulating glucose and lipid metabolism, and can also be anti-inflammatory,antioxidative, thus inhibiting atherosclerosis[12–15].PPARα can inhibit VSMCs proliferation and promote their apoptosis[18],and can also inhibit VSMCs proliferation by upregulating HO-1 expression, a target gene [22].Both HO-1 and CO can inhibit VSMCs proliferation[18][13].

However, whether the Irisin/PPARα signaling pathway can mediate HO-1 to regulate the balance between VSMCs proliferation and apoptosis, thereby preventing atherosclerosis formation and development is unclear. Therefore, it is important for theoretical research and clinical treatment to explore the role and molecular mechanism of Irisin/PPARα signaling pathway on regulation of the balance between proliferation and apoptosis of VSMCs, and to find a new target for the prevention and treatment of the occurrence and development of atherosclerosis.

In this study, it was found that overexpression of FNDC5 significantly increased PPARα, HO-1, Bax, caspase-3, and Bax/Bcl-2 mRNA and protein expression levels, and significantly increased the apoptosis rate of VSMCs; however, Bcl-2 mRNA and protein expression levels were significantly decreased, DNA synthesis in VSMCs was significantly reduced, and the proliferation activity and proliferation rate of VSMCs were significantly decreased.

In contrast, silencing FNDC5 significantly decreased PPARα, HO-1, Bax, caspase-3, and Bax/Bcl-2 mRNA and protein expression levels, and significantly reduced the apoptosis rate of VSMCs, while Bcl-2 mRNA and protein expression levels were significantly increased, DNA synthesis in VSMCs was significantly increased, and the proliferation activity and proliferation rate of VSMCs were significantly increased.Further addition of PPARα inhibitor GW6471 on the basis of overexpression of FNDC5 could down-regulate PPARα, HO-1, Bax, caspase-3, and Bax/Bcl-2 mRNA and protein expression in VSMCs, and compared with the overexpression FNDC5 group, DNA synthesis was significantly increased in VSMCs, and the proliferation activity and proliferation rate of VSMCs were significantly increased, while the apoptosis rate of VSMCs was significantly reduced.

Previous studies by our group have confirmed that irisin alleviates the formation of atherosclerosis through anti-inflammatory and anti-oxidative effects, and can reduce the levels of inflammatory mediators hs-CRP, NF-κB and MDA in patients with acute coronary syndrome and play a protective role in coronary heart disease[13, 14].One study[15] and the initial exploration of this study found that irisin could inhibit VSMCs proliferation and migration.However,the mechanism by which irisin inhibits the proliferation of VSMCs is unclear, and as far as we know, there are no reports on irisin regulating the apoptosis of VSMCs.PPARα can inhibit VSMCs proliferation and neointima formation after carotid balloon injury by promoting p16 expression, a cycle- dependent kinase inhibitor in VSMCs, and arresting cell cycle progression from G1 to S phase[28].Increased PPARα mRNA expression can significantly increase the rate of vascular smooth muscle cell apoptosis[29].PPARα overexpression inhibited ox-LDL-induced VSMCs proliferation and promoted their apoptosis[15].PPARα directly activates HO-1 gene expression by activating the HO-1 promoter through a fragment located between 1.4kb and 2.2kb from the transcription start site, thereby inhibiting VSMCs proliferation[22].Induction of HO-1 expression inhibits platelet-derived growth factor-induced proliferation of VSMCs[30].Transfection of rat aortic VSMCs with HO-1 overexpression adenovirus can promote their apoptosis, and the degree of apoptosis is positively correlated with the increased expression level of the pro-apoptotic protein P53, and administration of exogenous biliverdin or bilirubin can also promote the apoptosis of VSMCs [31].Previous studies by our group have confirmed that the HO-1/CO system is directly involved in the regulation of VSMCs proliferation by inhibiting cellular DNA synthesis, preventing cell cycle progression[21], and preventing aortic intimal thickening and neointima formation after carotid balloon injury by inhibiting the expression of inflammatory mediators NF-κB, TNF-α, and ET-1[21].In this study, overexpression of FNDC5 could significantly increase PPARα, HO-1, Bax, caspase-3, and Bax/Bcl-2 expression and promote VSMCs apoptosis; it also significantly decreased Bcl-2 expression and inhibited DNA synthesis, proliferation activity, and proliferation rate of vascular smooth muscle cells; silencing FNDC5 presented opposite results.Further addition of GW6471, a PPARα inhibitor, on the basis of overexpression of FNDC5, could down-regulate PPARα, HO-1, Bax, caspase-3, and Bax/Bcl-2 expression, and the DNA synthesis, proliferation activity, and compared with the overexpression FNDC5 group, proliferation rate of vascular smooth muscle cells were significantly increased, while the apoptosis rate was significantly decreased.It can be seen that the regulatory effect of irisin/PPARα signaling pathway on the proliferation of VSMCs showed the opposite trend to HO-1 expression level, while the regulatory effect on apoptosis showed the same trend as HO-1 expression level, indicating that HO-1 can be directly involved in the regulation of VSMCs proliferation by inhibiting VSMCs DNA synthesis, but also promote VSMCs apoptosis by regulating the expression of Bax, Bcl-2, caspase-3, and Bax/Bcl-2, the important functional molecules of apoptosis signaling pathway.HO-1, as an inducible heme oxygenase, is activated in pathological conditions, and its up-regulation is a protective response of cells against oxidative stress and injury.It was further shown that Irisin regulates the balance between proliferation and apoptosis of VSMCs through the targeted induction of HO-1 mRNA and protein expression by PPARα, which may be the mechanism of Irisin against atherosclerosis.

In summary, overexpression of FNDC5 can upregulate PPARα expression and then induce increased HO-1 expression, on the one hand, it prevents VSMCs proliferation by inhibiting VSMCs DNA synthesis;the other hand, it promotes the apoptosis of VSMCs by regulating the expression of Bax, Bcl-2, caspase-3, Bax/Bcl-2 mRNA and protein, which are important functional molecules of apoptosis signaling pathway;These results suggest that irisin/PPARα signaling pathway can mediate the balance of HO-1 regulating the proliferation and apoptosis of VSMCs, which may play an important role in the prevention and treatment of atherosclerosis and restenosis, therefore, irisin is expected to be a new target for the prevention and treatment of atherosclerosis and restenosis formation.

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No competing interests reported.

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Irisin/PPARá Mediates the Proliferation and Apoptosis of Ascular Smooth Muscle Cells through Activation of the HO-1

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Abstract

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Background

1. Materials And Methods

2. Results

2.1 Lentivirus-transfected VSMCs and selection of stable cell lines

Table 1

Comparison of proliferation activity of VSMCs in each group（`x±s）

3. Discussion

References

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