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NOS gene transfer inhibits expression of cell cycle regulatory molecules in vascular smooth muscle cells
1Department of Anatomy and Cell Biology and The Cardiovascular Center, The University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Abstract:
The mechanisms of nitric oxide (NO)-mediated inhibition of vascular smooth muscle (VSM) cell proliferation are still obscure. Cyclins A and E in association with cyclin-dependent kinase 2 (cdk2) serve as positive regulators for mammalian cell cycle progression through the G1/S checkpoint of the cell cycle and subsequent cell proliferation. Therefore, we have tested the effect of adenovirus-mediated transfection of the endothelial nitric oxide synthase (eNOS) gene into guinea pig coronary VSM cells on platelet-derived growth factor (BB homodimer) (PDGF-BB)-stimulated cell proliferation and the expression of cell cycle regulatory molecules. Transfection of the eNOS gene (eNOS) into VSM cells significantly inhibited (P < 0.05) [3H]thymidine incorporation into the DNA in response to PDGF-BB stimulation compared with lacZ-transfected control cells. The eNOS transfer significantly inhibited (P < 0.05) PDGF-BB-induced proliferating cell nuclear antigen (PCNA) and cyclin A expression in VSM cells compared with cells transfected with the control vector. The time course of cyclin E expression in response to PDGF-BB stimulation was delayed in eNOS-transfected cells. Levels of cyclin-dependent kinase inhibitors p21 and p27 were not significantly affected by eNOS transfer. eNOS transfer did not decrease PDGF-beta receptor number, affinity, and autophosphorylation measured by radioreceptor assay and Western analysis. These results suggest that inhibition of PDGF-stimulated expression of cyclin A, cyclin E, and PCNA is the target of NO action. These findings could explain, at least in part, NO-mediated inhibition of VSM cell proliferation.
Insights
Nitric oxide (NO) inhibits vascular smooth muscle cell proliferation by reducing cyclin A and E expression. This study shows endothelial nitric oxide synthase gene transfer blocks platelet-derived growth factor-stimulated cell growth.
Area of Science:
- Cardiovascular Biology
- Molecular Biology
- Cell Cycle Regulation
Background:
- Vascular smooth muscle (VSM) cell proliferation contributes to cardiovascular diseases.
- Nitric oxide (NO) is known to inhibit VSM cell proliferation, but the precise mechanisms remain unclear.
- Cyclins and cyclin-dependent kinases regulate cell cycle progression.
Purpose of the Study:
- To investigate the effect of endothelial nitric oxide synthase (eNOS) gene transfer on platelet-derived growth factor (PDGF-BB)-stimulated VSM cell proliferation.
- To determine the impact of eNOS gene transfer on the expression of key cell cycle regulatory molecules, including cyclins and cyclin-dependent kinase inhibitors.
Main Methods:
- Adenovirus-mediated transfection of the eNOS gene into guinea pig coronary VSM cells.
- Assessment of [3H]thymidine incorporation to measure DNA synthesis and cell proliferation.
- Western blot analysis and radioreceptor assays to evaluate the expression of proliferating cell nuclear antigen (PCNA), cyclins A and E, cyclin-dependent kinase inhibitors (p21, p27), and PDGF-beta receptor.
Main Results:
- eNOS gene transfer significantly inhibited PDGF-BB-stimulated DNA synthesis in VSM cells.
- eNOS transfer significantly reduced the expression of PCNA and cyclin A in response to PDGF-BB.
- PDGF-BB-induced cyclin E expression was delayed in eNOS-transfected cells, while p21 and p27 levels remained unaffected.
- eNOS transfer did not alter PDGF-beta receptor number, affinity, or autophosphorylation.
Conclusions:
- NO-mediated inhibition of VSM cell proliferation likely involves the suppression of PDGF-stimulated expression of cyclin A, cyclin E, and PCNA.
- These findings provide mechanistic insights into how NO regulates VSM cell proliferation, potentially offering therapeutic targets for cardiovascular diseases.