Evaluation of Cell Proliferation in Human Atherosclerotic Lesions

Abstract

Local accumulation of cells (hypercellularity) in the intima of the arterial wall is recognized as one of the major manifestations of human atherosclerosis, which may result from local cell proliferation (1-3). This assumption was raised both on the comparative analysis of cell number in nondiseased and atherosclerotic areas of human arteries (3-5) and on the basis of the analysis of cell replication in experimental atherosclerosis (for review see ref. 1). In experimental atherosclerosis, cell replication was identified in several models, including mechanical injury of the arteries and experimental hypercholesterolemia. The experimental approach permits the direct determination of DNA replication using radiolabeled precursor-[³H]-thymidine, but it is impossible to demonstrate proliferation in human vessels directly, using standard procedures with labeling of replicating DNA. Villaschi and coworkers (6) labeled human arteries with [³H]-thymidine-ex vivo. They found a low level of replication in both grossly normal tissues and atherosclerotic plaques. In the case of ex vivo labeling, the questions of diffusion and of thymidine incorporation into ex vivo incubated tissue (6) make it difficult to be sure that this procedure reflects replication rates in vivo. Use of proliferation-specific antibodies is advantageous, since proliferating cells are detected directly. Antibodies to the cell-cycle related protein, PCNA (cyclin), are now commonly used in the study of human tissues (7-12). PCNA is an auxiliary protein of mammalian DNA polymerase-δ and is a stable cell-cycle-regulated nuclear protein (synthesized mainly in the S-phase but also present in G1 and G2 phases of the cell cycle), the presence of which correlates directly with a proliferative state of normal cells (7-9).