Modified quantitative detection of apoptosis in coronary atherosclerosis

Apoptosis, or programmed cell death, is a physiological cell death process that allows a tissue or organ to remove unwanted cells and responsible for the maintenance of stable cell numbers in tissues even in primary atherosclerotic or restenotic lesion of coronary artery. Previous studies on the apoptosis of coronary atherosclerosis are usually prone to run into qualitative description without the aid of "quantitative" evaluation. To eliminate such subjective errors, we have designed a triple immunofluorescent stain assessed by digital camera and computer-aided analysis system so that the actual quantitative apoptotic expression of coronary atherosclerotic development can be more precisely evaluated.

Specimens of coronary artery were obtained from consecutive patients undergoing coronary endarterectomy or cardiac transplantation. Twenty-seven blocks of paraffin tissue specimens from 16 patients were analyzed. According to the American Heart Association (AHA) classification of atherosclerotic lesion, type I, II, III lesions were defined as early lesions and the other three types: IV, V, and VI as advanced lesions. Apoptosis and cell types were recognized simultaneously by triple immunofluorescent stain combined with Hoechst, Terminal deoxynucleotidyl transferase-mediated dUTP Nick End-Labeling (TUNEL) and smooth muscle cell (SMC) marker staining. Apoptotic index deduced from TUNEL-positive nucleus number divided by Hoechst-positive nucleus number. Apoptotic cell-type index deduced from TUNEL-positive nuclei, which are surrounded by cell type fluorescent marker, divided by total TUNEL-positive nuclei.

Fourteen early lesions and 13 advanced lesions of tissue specimens were analyzed in this study. The mean apoptotic index of early lesions was 2.60 +/- 1.72%, which is significantly lower than that of the advanced lesions (7.42 +/- 3.07%). The apoptosis between the tapering portion and lesion portion obtained from 10 coronary endarterectomy specimens was also significantly different (2.59 +/- 1.90% vs 8.10 +/- 3.20%). In either early or advanced lesions, the predominant cell type of apoptosis was SMC.

The identical counting and quantification analytic method we designed is more accurate and quantitative than the traditional investigation in detecting and affirming the "homeostasis" role of apoptosis in the atherosclerotic pathogenesis process of coronary artery disease.