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Updated: Aug 19, 2026
Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function
Published on: June 11, 2017
Altered AP-1/Ref-1 redox pathway and reduced proliferative response in iNOS-deficient vascular smooth muscle cells
Kuang-Yuh Chyu1, Paul C Dimayuga, Xiaoning Zhao
1Atherosclerosis Research Center, Burns and Allen Research Institute, Division of Cardiology, Cedars-Siffai Medical Center/David Geffen School of Medicine, UCLA, Los Angeles, CA 90048, USA. Chyuk@cshs.org
Abstract:
We previously reported that injury-induced medial vascular smooth muscle cell (VSMC) proliferation and neointima formation in carotid arteries of inducible nitric oxide synthase knockout (iNOS KO) mice were significantly reduced compared with wild type (WT). However, the molecular pathway underlying such differences is not known. In this in vitro study, we discovered that the AP-1/Ref-1/thioredoxin signaling pathway is altered in aortic VSMC from iNOS KO mice, which leads to reduced growth response when compared with aortic VSMC from WT mice. After equal initial seeding, the cell number after 7 days in serum medium was less in iNOS KO cells compared with WT VSMC (1.2 +/- 0.6 x 10(5) vs 3.2 +/- 1.1 x 10(5); p < 0.05). Significantly more iNOS KO cells remained in the G0/G1 phase compared with WT cells after 24-h serum treatment (82.6 +/- 13.7% vs 62.3 +/- 14.6%; p < 0.05) by cell-cycle analysis. Nuclear PCNA expression was also less in the iNOS KO cells, which was not affected by exogenous NO or superoxide. Superoxide generation after 24-h serum stimulation was less in the iNOS KO cells compared with WT cells. After 30-min serum stimulation, AP-1 DNA binding was reduced and a lack of increase in nuclear c-Jun protein was observed in iNOS KO VSMC. RT-PCR analysis confirmed a lack of inducible c-Jun mRNA after serum stimulation in the KO cells. In addition, KO cells had less nuclear reducing factor-1 (Ref-1) and serum-inducible thioredoxin protein expression. Reduced proliferative response of iNOS KO VSMC to serum treatment is associated with altered AP-1 /Ref-1 /thioredoxin pathway activation.
Insights
Vascular smooth muscle cell proliferation is reduced in inducible nitric oxide synthase knockout mice due to an altered AP-1/Ref-1/thioredoxin pathway, impacting neointima formation.
Area of Science:
- Vascular Biology
- Cell Signaling
- Molecular Medicine
Background:
- Injury-induced vascular smooth muscle cell (VSMC) proliferation and neointima formation are critical in cardiovascular diseases.
- Previous studies showed reduced neointima formation in inducible nitric oxide synthase knockout (iNOS KO) mice.
- The underlying molecular mechanisms for these differences remain unclear.
Purpose of the Study:
- To investigate the molecular pathway responsible for reduced VSMC proliferation in iNOS KO mice.
- To compare the growth response of VSMC from iNOS KO and wild-type (WT) mice in vitro.
- To elucidate the role of the AP-1/Ref-1/thioredoxin signaling pathway in VSMC proliferation.
Main Methods:
- In vitro study using aortic VSMC from iNOS KO and WT mice.
- Cell proliferation assays (cell counting, cell-cycle analysis).
- Analysis of signaling pathway components including AP-1 DNA binding, c-Jun expression, Ref-1, and thioredoxin.
Main Results:
- iNOS KO VSMC exhibited significantly reduced proliferation compared to WT VSMC after serum stimulation.
- iNOS KO cells showed a higher G0/G1 phase arrest and reduced nuclear PCNA expression.
- Altered activation of the AP-1/Ref-1/thioredoxin pathway was observed in iNOS KO VSMC, with reduced AP-1 DNA binding, c-Jun induction, Ref-1, and thioredoxin expression.
Conclusions:
- The AP-1/Ref-1/thioredoxin signaling pathway is significantly altered in iNOS KO VSMC.
- This altered pathway contributes to the reduced proliferative response of VSMC from iNOS KO mice.
- Findings suggest a novel molecular mechanism linking iNOS to VSMC growth regulation and neointima formation.
