Angiotensin Ⅱ type 1 receptor autoantibody-AT1R-Bmal1 axis promotes phenotypic transition of vascular smooth muscle cells and vascular fibrosis

XUE Lingxia; LONG Yaolin; FENG Jiayan; MAO Tian; GUO Jiao; WANG Zhuoxi; LI Yang; WANG Xiaohui; WANG Li

doi:10.16016/j.2097-0927.202412142

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Angiotensin Ⅱ type 1 receptor autoantibody-AT1R-Bmal1 axis promotes phenotypic transition of vascular smooth muscle cells and vascular fibrosis

Basic Medicine | 更新时间：2025-06-11

- Angiotensin Ⅱ type 1 receptor autoantibody-AT1R-Bmal1 axis promotes phenotypic transition of vascular smooth muscle cells and vascular fibrosis
- Journal of Army Medical University Vol. 47, Issue 11, Pages: 1155-1164(2025)
- 作者机构：
  
  山西医科大学基础医学院，山西太原
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(31871177);the Youth Scientific Research Project of Shanxi Basic Research Programme(202203021212360);the Science and Technology Innovation Project of Higher Education Institutions in Shanxi Province(2022L115)
- DOI：10.16016/j.2097-0927.202412142
  CLC： R322.12;R329.26;R392.11
- Received：29 December 2024，
  
  Revised：23 March 2025，
  
  Published：15 June 2025
- 稿件说明：
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薛灵霞，龙瑶琳，冯佳艳，等.血管紧张素Ⅱ-1型受体自身抗体-AT1R-Bmal1轴促进血管平滑肌细胞表型转换和血管纤维化［J］.陆军军医大学学报，2025，47（11）：1155-1164.DOI：10.16016/j.2097-0927.202412142.

XUE L X，LONG Y L，FENG J Y，et al.Angiotensin Ⅱ type 1 receptor autoantibody-AT1R-Bmal1 axis promotes phenotypic transition of vascular smooth muscle cells and vascular fibrosis［J］.J Army Med Univ，2025，47（11）：1155-1164.DOI：10.16016/j.2097-0927.202412142.
薛灵霞，龙瑶琳，冯佳艳，等.血管紧张素Ⅱ-1型受体自身抗体-AT1R-Bmal1轴促进血管平滑肌细胞表型转换和血管纤维化［J］.陆军军医大学学报，2025，47（11）：1155-1164.DOI：10.16016/j.2097-0927.202412142. DOI：

XUE L X，LONG Y L，FENG J Y，et al.Angiotensin Ⅱ type 1 receptor autoantibody-AT1R-Bmal1 axis promotes phenotypic transition of vascular smooth muscle cells and vascular fibrosis［J］.J Army Med Univ，2025，47（11）：1155-1164.DOI：10.16016/j.2097-0927.202412142. DOI：

摘要

目的

探讨血管紧张素Ⅱ-1型受体（angiotensin Ⅱ type 1 receptor， AT1R）自身抗体（AT1R autoantibody， AT1-AA）通过促进血管平滑肌细胞（vascular smooth muscle cells， VSMCs）生物钟蛋白BMAL1异常表达诱导细胞表型转换以及血管纤维化的作用机制。

方法

将12只6~8周的SD雄性大鼠（体质量180~220 g）按随机数字表法分为2组（

=6）：对照组以及使用AT1R细胞外第二环（extracellular loop Ⅱ of angiotensin Ⅱ type 1 recep

tor， AT1R-ECLⅡ）主动免疫SD大鼠建立的AT1-AA阳性组。采用HE染色和Masson染色分别观察2组大鼠胸主动脉血管结构变化和纤维化情况（

=3）。采用Western blot检测血管组织以及原代VSMCs中纤维化标志蛋白Collagen Ⅰ、表型转换相关蛋白SM22、α-SMA、OPN以及MMP2的表达（

=4），此外，还检测了CT0、CT4、CT8、CT12、CT16、CT20时生物钟蛋白BMAL1的表达。通过Transwell实验和划痕实验检测VSMCs的增殖迁移能力（

=3），使用si-RNA技术敲低Bmal1后检测BMAL1、Collagen Ⅰ、表型转换相关蛋白的表达（

=3）。另外，构建AT1-AA阳性的AT1R敲除（AT1R-KO）大鼠模型使用Western blot检测其胸主动脉血管组织BMAL1的表达（

=4）。

结果

AT1-AA阳性大鼠胸主动脉血管壁增厚［（140±9）％

（120±5）%，

0.05］，血管平滑肌细胞排列紊乱，Masson染色蓝染明显，且Collagen Ⅰ表达较Control组表达上调（

0.05）。AT1-AA阳性大鼠胸主动脉以及AT1-AA处理的VSMCs中收缩表型相关蛋白α-SMA、SM22表达下降（

0.05），合成表型相关蛋白OPN、MMP2表达上调（

0.05）；AT1-AA诱导VSMCs划痕愈合能力以及迁移能力都显著增强。进一步发现，Bmal1的mRNA表达（0.67±0.26

1.03±0.17）以及蛋白表达（0.46±0.06

0.85±0.26）均在CT12时显著上调（

0.05），且Bmal1的节律性消失（

0.05）。敲低BMAL1后，AT1-AA诱导的VSMCs表型转换现象得到部分改善。与AT1-AA阳性的WT大鼠比较，AT1-AA阳性的AT1R-KO大鼠胸主动脉血管中BMAL1表达明显降低（1.35±0.06

0.86±0.07，

0.001）；细胞水平发现AT1-AA诱导的VSMCs表型转换及胶原蛋白Collagen Ⅰ高表达的现象在AT1R-KO的VSMCs中得到部分改善。

结论

AT1-AA通过AT1R-Bmal1轴促进VSMCs表型转换以及血管纤维化。

Abstract

Objective‍　‍To investigate the mechanism by which angiotensin Ⅱ type 1 receptor autoantibody (AT1-AA) promotes phenotypic switch of vascular smooth muscle cells (VSMCs) and vascular fibrosis through abnormal expression of circadian clock protein BMAL1. Methods‍　‍Twelve male SD rats (6~8 weeks old

weighing 180~220 g) were randomly divided into (

=6) a control group and an AT1-AA-positive group [established by active immunization of SD rats with AT1R extracellular loop Ⅱ peptide (AT1R-ECLⅡ)

]

. HE and Masson stainings were used to observe structural changes and fibrosis in the thoracic aorta (

=3). Western blotting

was performed to detect the expression of Collagen I

phenotypic switch-related proteins (SM22

α-SMA

OPN and MMP2) in vascular tissues and primary VSMCs (

=4)

as well as the expression of BMAL1 at CT0

CT4

CT8

CT12

CT16

and CT20. Transwell and scratch assays were used to assess the proliferation and migration of VSMCs (

=3). si-RNA was employed to knock down Bmal1

followed by detection of BMAL1

Collagen I

and phenotypic conversion-related protein expression (

=3). Additionally

AT1-AA-positive AT1R-knockout (AT1R-KO) rats were constructed to measure BMAL1 expression in thoracic aortic tissues (

=4). Results‍　‍The AT1-AA-positive rats had significantly thickened thoracic aortic vessel wall [(140±9)%

(120±5)%

0.05

]

badly arranged VSMCs

obvious blue Masson staining

and up-regulated Collagen I expression (

0.05). In the thoracic aorta of AT1-AA-positive rats and AT1-AA-treated VSMCs

the expression of contractile phenotype-related proteins (α-SMA

SM22) was decreased (

0.05)

while the expression of synthetic phenotype-related proteins (OPN

MMP2) was increased (

0.05). AT1-AA enhanced the scratch healing ability and migration ability of VSMCs. Furthermore

both mRNA and protein levels of Bmal1 were significantly up-regulated at CT12 (

0.05)

and the rhythmicity of Bmal1 was lost. Knockdown of Bmal1 partially ameliorated AT1-AA-induced phenotypic switch of VSMCs. Compared with AT1-AA-positive WT rats

AT1-AA-positive AT1R-KO rats showed significantly reduced BMAL1 expression in the thoracic aorta (1.35±0.06

0.86±0.07，

0.001). At the cellular level

AT1-AA-induced phenotypic switch and high Collagen I expression in VSMCs were partially improved in AT1R-KO VSMCs. Conclusion‍　‍AT1-AA promotes VSMCs phenotypic conversion and vascular fibrosis through the AT1R-Bmal1 axis.

关键词

Keywords

references

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