Molecular characterization of rabbit CPP32 and its function in vascular smooth muscle cell apoptosis

Area of Science:

• Cardiovascular Biology
• Cellular Biology
• Molecular Medicine

Background:

• Vascular remodeling in atherogenesis involves cellular proliferation, migration, and apoptosis.
• Caspase proteases, particularly CPP32, are central to apoptosis pathways.
• Nitric oxide (NO) donors like sodium nitroprusside (SNP) can induce apoptosis in vascular smooth muscle cells (SMC).

Purpose of the Study:

• To investigate the role of CPP32 in SNP-induced apoptosis of SMC.
• To determine if CPP32 activation or expression changes contribute to NO-mediated SMC cell death.

Main Methods:

• Isolated rabbit CPP32 cDNA via PCR.
• Measured CPP32 protease activity using DEVD-pNA substrate.
• Performed immunoblot analysis for CPP32 cleavage and PARP degradation.
• Assessed DNA fragmentation using ELISA, in situ end labeling, and gel electrophoresis.
• Utilized a CPP32 inhibitor (DEVD-CHO) to evaluate its effect on apoptosis.

Main Results:

• SNP treatment significantly increased CPP32 protease activity in SMC (up to 10-fold).
• SNP induced CPP32 cleavage into its active p17 subunit and PARP degradation.
• Inhibition of CPP32 with DEVD-CHO reduced SNP-induced DNA fragmentation by 50%.
• CPP32 mRNA levels remained unchanged after SNP treatment, indicating post-transcriptional regulation.

Conclusions:

• CPP32 mRNA is constitutively expressed in rabbit SMC.
• Activation of CPP32 protein, not increased mRNA levels, is crucial for SNP-induced SMC apoptosis.
• CPP32 plays a pivotal role in the apoptotic cell death of vascular smooth muscle cells stimulated by NO donors.