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Updated: Aug 24, 2026
Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice
Published on: February 20, 2019
Quiescence, cell viability, apoptosis and necrosis of smooth muscle cells using different growth inhibitors
J Pelisek1, S Armeanu, S Nikol
1Medical Clinic I, Klinikum Grosshadern, Ludwig Maximilian University, Munich, Germany.
Abstract:
Smooth muscle cells and endothelial cells play an important role in cardiovascular diseases and may therefore be a potential target for gene therapy. Most in vitro experiments are performed using proliferating cell cultures. Nevertheless, non-dividing cells would represent more realistic in vivo conditions for gene therapy. Therefore, a simple method to achieve physiologically quiescence in cell cultures is needed for experiments. Growth to confluence is sufficient for endothelial cells to reach quiescence, in contrast to smooth muscle cells. Alternative techniques were investigated to achieve quiescence for smooth muscle cells. N-acetyl-cysteine, heparin, aphidicolin and serum-free medium are known inhibitors of smooth muscle cell proliferation and were tested for cell viability, necrosis and apoptosis. The inhibition status was evaluated counting cells in a cell counter. Toxicity, necrosis and apoptosis were determined using FACS analysis. Then, smooth muscle cells and endothelial cells were transfected with plasmid containing the beta-galactosidase gene using liposomes. Analysis of gene expression in transfected cells included a quantitative beta-galactosidase assay and X-gal staining. Growth inhibition was achieved with all agents tested. Using N-acetyl-cysteine, only slightly reduced growth rates were observed. Aphidicolin stopped cell growth almost immediately, but demonstrated enhanced toxicity. The amount of apoptotic and necrotic cells was lowest using heparin in the presence of foetal calf serum. Transfection experiments using stationary cultures of smooth muscle cells using heparin or aphidicolin demonstrated 5-10-fold lower transfection rates compared to transfected proliferating cell cultures serving as controls. Transfection experiments using stationary cultures of endothelial cells using growth inhibition through confluence demonstrated 40-fold lower transfection rates than transfected proliferating cell cultures. Transfer efficiency was much lower in endothelial cells compared to smooth muscle cells. In conclusion, quiescent cells simulate more realistically the in vivo situation and may therefore represent a better model for future in vivo experiments based on in vitro findings.
Insights
Achieving quiescent cells in vitro is crucial for realistic gene therapy models. Heparin effectively induced quiescence in smooth muscle cells with minimal toxicity, though transfection rates remained lower than in proliferating cells.
Area of Science:
- Cardiovascular Research
- Gene Therapy
- Cell Biology
Background:
- Smooth muscle cells (SMCs) and endothelial cells (ECs) are key in cardiovascular diseases and gene therapy targets.
- In vitro gene therapy studies often use proliferating cells, not reflecting in vivo conditions.
- Quiescent (non-dividing) cells offer a more realistic model for in vivo gene therapy research.
Purpose of the Study:
- To develop a simple method for inducing physiological quiescence in SMCs and ECs for in vitro gene therapy experiments.
- To evaluate the efficacy and toxicity of different agents in achieving SMC quiescence.
- To compare gene transfer efficiency in quiescent versus proliferating SMCs and ECs.
Main Methods:
- SMCs and ECs were treated with N-acetyl-cysteine, heparin, aphidicolin, or serum-free medium to induce quiescence.
- Cell viability, necrosis, and apoptosis were assessed using FACS analysis.
- Gene transfection was performed using liposomes and a beta-galactosidase reporter gene.
- Transfection efficiency was quantified via beta-galactosidase assay and X-gal staining.
Main Results:
- All tested agents inhibited SMC proliferation; aphidicolin showed high toxicity, while heparin resulted in the lowest apoptosis and necrosis.
- Heparin or aphidicolin treatment led to 5-10 fold lower transfection rates in quiescent SMCs compared to proliferating controls.
- Confluence-induced quiescence in ECs resulted in a 40-fold decrease in transfection rates compared to proliferating ECs.
- Overall gene transfer efficiency was significantly lower in quiescent cells, especially in ECs.
Conclusions:
- Quiescent cells provide a more accurate in vivo simulation for gene therapy research.
- Heparin is a promising agent for inducing SMC quiescence with acceptable cell viability.
- Further optimization is needed to improve gene transfer efficiency in quiescent cells for effective in vivo gene therapy applications.
