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Mitochondrial release of apoptosis-inducing factor and cytochrome c during smooth muscle cell apoptosis
D J Granville1, B A Cassidy, D O Ruehlmann
1Department of Pathology and Laboratory Medicine, University of British Columbia McDonald Research Laboratories/The iCAPTURE Centre, St. Paul's Hospital/Providence Health Care, University of British Columbia, Vancouver, British Columbia, Canada.
Abstract:
Photodynamic therapy (PDT) is under investigation for the treatment of intimal hyperplastia in conditions such as atherosclerosis and restenosis. Although smooth muscle cells (SMCs) may be a key target for treatment, the effects of PDT on these cells are poorly characterized. In the present study, apoptosis was induced in primary human aortic SMCs by the combination of the photosensitizer verteporfin and visible light. After PDT, an increase in mitochondrial cytochrome c (cyt c) and apoptosis-inducing factor (AIF) levels were detected in the cytosol immediately and their levels increased steadily up to 2 hours. Cytosolic levels of the pro-apoptotic Bcl-2 family member Bax decreased reciprocally throughout this period, but this change did not occur before cyt c release. Confocal microscopy revealed a diffuse staining pattern of cyt c within apoptotic cells as compared to a distinct mitochondrial staining in normal cells. AIF translocated from mitochondria to the nucleus during the progression of apoptosis. After cyt c release, caspase-9 and caspase-3 processing was visible by 1 hour and caspase-6, -7, and -8 processing was apparent by 2 hours after PDT. In summary, these results demonstrate for the first time the cellular redistribution of mitochondrial AIF during SMC apoptosis, as well as the early release of cyt c and the subsequent activation of multiple caspases during PDT-induced SMC apoptosis.
Insights
Photodynamic therapy (PDT) induces apoptosis in human aortic smooth muscle cells (SMCs) by releasing cytochrome c and apoptosis-inducing factor. This study details the molecular events, including caspase activation, during PDT-induced SMC apoptosis.
Area of Science:
- Biomedical Engineering
- Cell Biology
- Molecular Medicine
Background:
- Photodynamic therapy (PDT) shows promise for treating intimal hyperplasia in atherosclerosis and restenosis.
- Smooth muscle cells (SMCs) are a potential therapeutic target in these conditions, but PDT's effects on SMCs are not well understood.
Purpose of the Study:
- To investigate the mechanisms of apoptosis induced by PDT in primary human aortic SMCs.
- To characterize the cellular redistribution of key apoptotic factors like cytochrome c and AIF.
- To determine the role of caspases in PDT-induced SMC apoptosis.
Main Methods:
- Primary human aortic SMCs were treated with verteporfin and visible light to induce apoptosis.
- Mitochondrial and cytosolic levels of cytochrome c (cyt c) and apoptosis-inducing factor (AIF) were measured.
- Confocal microscopy was used to track the cellular localization of cyt c and AIF.
- Caspase processing was analyzed to assess apoptotic pathway activation.
Main Results:
- PDT induced apoptosis in SMCs, characterized by the release of mitochondrial cyt c and AIF into the cytosol.
- AIF translocated from mitochondria to the nucleus, while cyt c staining became diffuse in apoptotic cells.
- The pro-apoptotic protein Bax levels decreased after cyt c release.
- Multiple caspases (caspase-3, -6, -7, -8, -9) were activated following PDT.
Conclusions:
- PDT triggers SMC apoptosis through the release of mitochondrial factors and subsequent caspase activation.
- This study provides the first evidence of AIF redistribution during PDT-induced SMC apoptosis.
- The findings elucidate the molecular cascade of PDT-induced SMC apoptosis, relevant for therapeutic applications in vascular diseases.