Related Experiment Video
Updated: Jul 18, 2026
Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro
Published on: February 15, 2022
Proteomic profiling and identification of cofilin responding to oxidative stress in vascular smooth muscle
Chang-Kwon Lee1, Hyo-Jun Park, Hyeon Ha So
1Department of Physiology, Konkuk University, Danwol-dong, Chungju City, Chungbuk, Korea.
Abstract:
We used 2-DE and MALDI-TOF/TOF to identify proteins of vascular smooth muscle cells whose expression was or was not altered by exposure to 500 microM H2O2 for 30 min. We detected more than 800 proteins on silver-stained gels of whole protein extracts from rat aortic smooth muscle strips. Of these proteins, 135 clearly unaffected and 19 having levels altered by exposure to H2O2 were identified. Protein characterization revealed that the most prominent vascular smooth muscle proteins were those with antioxidant, cytoskeletal structure, or muscle contraction. In addition, cofilin, an isoform of the actin depolymerizing factor family, shifted to its basic site on the 2-DE gel as a result of H2O2 treatment. In Western blot analysis of proteins from A7r5 aortic smooth muscle cells, the phosphorylation, but not the expression, of cofilin was decreased by H2O2 in a dose-dependent manner. The H2O2-induced dephosphorylation of cofilin and apoptosis was inhibited by Na3VO4, an inhibitor of protein tyrosine phosphatase (PTP). These results suggest that cofilin is one of the proteins regulated by H2O2 treatment in vascular smooth muscle, and has an important role in the induction of vascular apoptosis through PTP-dependent mechanisms.
Insights
Hydrogen peroxide (H2O2) alters protein expression in vascular smooth muscle cells, notably affecting cofilin phosphorylation. This suggests cofilin plays a role in H2O2-induced vascular apoptosis via protein tyrosine phosphatase pathways.
Area of Science:
- Biochemistry
- Cell Biology
- Vascular Biology
Background:
- Oxidative stress, induced by hydrogen peroxide (H2O2), impacts cellular functions.
- Vascular smooth muscle cells (VSMCs) are crucial for blood vessel integrity and function.
- Understanding protein expression changes in VSMCs under oxidative stress is vital for cardiovascular research.
Purpose of the Study:
- To identify proteins in VSMCs affected by H2O2 exposure.
- To investigate the role of cofilin in H2O2-induced cellular responses.
- To elucidate the mechanisms underlying H2O2-mediated vascular smooth muscle cell apoptosis.
Main Methods:
- Two-dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF mass spectrometry for protein identification.
- Western blot analysis to assess protein phosphorylation and expression levels.
- Treatment with sodium orthovanadate (Na3VO4) to inhibit protein tyrosine phosphatase (PTP) activity.
Main Results:
- Over 800 proteins were detected in rat aortic smooth muscle cells; 19 showed altered levels upon H2O2 exposure.
- Cofilin, an actin-depolymerizing factor, exhibited altered migration on 2-DE gels and decreased phosphorylation in response to H2O2.
- H2O2-induced dephosphorylation of cofilin and apoptosis were mitigated by PTP inhibition.
Conclusions:
- Cofilin is a key protein regulated by H2O2 in vascular smooth muscle.
- H2O2-induced vascular smooth muscle cell apoptosis involves PTP-dependent mechanisms regulating cofilin.
- These findings highlight cofilin as a potential therapeutic target in oxidative stress-related vascular diseases.
