Targeting the E3 ligase RLIM to regulate VSMC phenotypic switching in vascular aging: implications for cold stress

Wenqian Jiang; Madi Guo; Xu Wang; Xin Liu; Yong Zhang

doi:10.1515/fzm-2025-0018

Targeting the E3 ligase RLIM to regulate VSMC phenotypic switching in vascular aging: implications for cold stress

doi: 10.1515/fzm-2025-0018

Wenqian Jiang^{1, 2, 3},
Madi Guo^{1, 2, 3},
Xu Wang^{1, 2, 3},
Xin Liu^{1, 2, 3},
Yong Zhang^{1, 2, 3
, ,}

1.
State Key Laboratory of Frigid Zone Cardiovascular Diseases (SKLFZCD), Department of Pharmacology, College of Pharmacy, and Department of Cardiology, the Second Affiliated Hospital, Harbin Medical University, Harbin 150081, China
2.
State Key Laboratory -Province Key Laboratories of Biomedicine-Pharmaceutics of China, and Key Laboratory of Cardiovascular Research, Ministry of Education, College of Pharmacy, Harbin 150081, China
3.
Research Unit of Noninfectious Chronic Diseases in Frigid Zone (2019RU070), Chinese Academy of Medical Sciences, Harbin 150081, China

Funds:

the National Natural Science Foundation of China 82273919

the HMU Marshal Initiative Funding HMUMIF-21022

More Information

Corresponding author: Yong Zhang, E-mail: hmuzhangyong@hotmail.com

摘要

Abstract: Objective Cold exposure may impair vascular function and promote cardiovascular diseases (CVDs) by causing vasoconstriction, hemodynamic changes, and sympathetic activation. Vascular aging, a key factor in CVDs, is linked to phenotypic switching of vascular smooth muscle cells (VSMCs), but its regulatory mechanisms are not fully understood. Materials and methods We used aged C57BL/6 mice and D-galactose-induced senescent VSMCs to investigate the role of the E3 ligase RLIM in arterial aging. RLIM knockdown and overexpression in vivo were achieved using adenoassociated virus (AAV) vectors. Vascular aging and stiffness were assessed using β-galactosidase staining, pulse wave velocity (PWV) measurements, and histological staining. Proteomic profiling was conducted to identify key protein alterations associated with vascular dysfunction and to elucidate underlying mechanisms. Results RLIM expression was significantly upregulated in the aortae of aged mice and D-galactose-induced senescent VSMCs. AAV-mediated RLIM knockdown significantly attenuated vascular aging, as evidenced by vascular ultrasound and histological assessments. Conversely, RLIM overexpression exacerbated vascular damage. Proteomic analysis revealed that RLIM knockdown in VSMCs from aged mice resulted in increased expression of smooth muscle contractile proteins and decreased levels of inflammatory markers, indicating a phenotypic shift toward a more contractile state. Conclusion These findings identify RLIM as a key regulator of arterial aging and a promising therapeutic target for age-related cardiovascular diseases.
- arterial aging /
- vascular remodeling /
- ubiquitination /
- vascular smooth muscle cell /
- phenotypic switching

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Figure 1. Elevated expression of ring finger protein-LIM domain interacting (RLIM) in aortic tissues from aged mice

(A) Venn diagram showing six differentially expressed E3 ligases in aortic vascular smooth muscle cells (VSMCs) from young and aged mice based on Tabula Muris Senis single-cell RNA-seq data. (B) qRT-PCR analysis of selected E3 ligases in aortae of young and aged mice. (C) Western blot analysis of RLIM protein levels in aortic tissues. (D) Immunohistochemistry showing increased RLIM expression in the medial layer of aged aortic sections. Scale bars, 50 μm. Statistical differences were ana-lyzed using an unpaired Student's t-test. N = 6 per group. Data are expressed as mean ± SD. ^***P < 0.001.

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Figure 2. Upregulation of ring finger protein-LIM domain interacting (RLIM) in D-galactose-induced senescence of mouse vascular smooth muscle cells (MOVAS) cells

(A) SA-β-gal staining of MOVAS cells treated with 0, 5, 25, or 50 mmol/L D-galactose for 12, 24, 36, or 48 hours. Quantification of SA-β-gal-positive cells is shown in the line graph. Scale bars, 50 μm. (B) Cell counting kit-8 (CCK-8) assay at different D-galactose concentrations and time points. (C) EdU incorporation assay assessing cell proliferation in control and D-galactose-treated MOVAS cells. Scale bars, 50 μm. (D) Western blot analysis of RLIM, p21, and p16 expression in MOVAS cells treat-ed with D-galactose compared to control. Statistical comparisons were performed using unpaired Student's t-test. N = 3-5 per group. Data are expressed as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

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Figure 3. Effects of ring finger protein-LIM domain interacting (RLIM) knockdown on vascular aging in young and aged mice

(A) Experimental timeline for adenoassociated virus (AAV)9-SM22α-shRLIM injection in young (3-month) and aged (18-month) mice. (B) SA-β-gal staining of aortae from young and aged mice treated with AAV9-SM22α-shRLIM or vector control. (C) Pulse wave velocity (PWV) measurements in the aortic arch and left common carotid artery of young and aged mice following RLIM knockdown in vascular smooth muscle cells (VSMCs). (D) Treadmill performance in young and aged mice with or without RLIM knockdown in VSMCs. (E) Histological images of aortic sections stained with hematoxylin and eosin (H & E), elastica-van-gieson (EVG), and Masson's trichrome in RLIM knockdown and control mice. Scale bars, 50 μm. Statistical analysis was performed using unpaired Student's t-test. N = 6 per group. Data are pre-sented as mean ± SD. ^*P < 0.017, ^**P < 0.003, ^***P < 0.0003 after Bonferroni correction.

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Figure 4. Effects of ring finger protein-LIM domain interacting (RLIM) overexpression on vascular aging and function in young and aged mice

(A) Experimental timeline of adenoassociated virus (AAV)9-SM22α-RLIM injections in young (3-month) and aged (18-month) mice. (B) Representative SA-β-gal stain-ing of aortic tissues from young and aged mice treated with AAV9-SM22α-RLIM or vector control. (C) Pulse wave velocity (PWV) measurements in the aortic arch and left common carotid artery of young and aged mice with or without RLIM overexpression in vascular smooth muscle cells (VSMCs). (D) Treadmill performance in young and aged mice following RLIM overexpression in VSMCs. (E) Histological staining hematoxylin and eosin (H & E), elastica-Van-Gieson (EVG), Masson's trichrome of aortic sections in RLIM-overexpressing and control mice. Scale bars, 50 μm. Statistical analysis was performed using unpaired Student's t-test. N = 6 per group. Data are expressed as mean ± SD. ^*P < 0.017, ^**P < 0.003, ^***P < 0.0003 after Bonferroni correction for multiple comparisons.

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Figure 5. Proteomic analysis of aortic tissue from aged mice with vascular smooth muscle cell (VSMC)-specific ring finger protein-LIM domain interacting (RLIM) knockdown

(A-B) Heatmap (A) and volcano plot (B) showing differentially expressed proteins (DEPs) in the aortae of aged mice treated with adenoassociated virus (AAV)9-SM22α-shRLIM compared to control mice, based on P < 0.05 and |log₂ fold change| ≥ 0.585. (C) Kyoto encyclopedia of genes and genomes (KEGG) pathway enrich-ment analysis of DEPs. Bubble size indicates the number of proteins involved in each pathway; color gradient represents statistical significance. (D) Heatmap of DEPs involved in smooth muscle contraction, cell cycle regulation, autophagy, and inflammation. (E) Venn diagram illustrating the overlap between DEPs and predicted RLIM ubiquitination substrates from the Ubibrowser 2.0 database. (F) Immunohistochemical staining of CSRP2 in aortic sections from young and aged mice with or without RLIM knockdown in VSMCs. Scale bars, 50 μm. Statistical analysis was performed using unpaired Student's t-test. N = 6 per group. Data are expressed as mean ± SD. ^*P < 0.017, ^**P < 0.003, ^***P < 0.0003 after Bonferroni correction.

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