Proliferative activity in peripheral and coronary atherosclerotic plaque among patients undergoing percutaneous revascularization.

We evaluated the proliferative activity of human atherosclerotic lesions associated with active symptoms of ischemia, by assessing the expression of the proliferating cell nuclear antigen (PCNA). We confirmed in vitro that PCNA, an essential component of the DNA synthesis machinery, is selectively expressed in proliferating human vascular smooth muscle cells. 37 atherosclerotic lesions (18 primary and 19 restenotic) retrieved by directional atherectomy from either coronary or peripheral arteries were then studied for the expression of PCNA, using in situ hybridization or immunohistochemistry. Among plaques studied by in situ hybridization, 7 out of 11 primary and 11 out of 11 restenotic lesions contained PCNA-positive cells. The mean rate of proliferation (percent of PCNA-positive cells) was 7.2 +/- 10.8% in primary lesions and 20.6 +/- 18.2% in restenotic lesions (P < 0.05). Among specimens studied by immunohistochemistry, five out of seven primary and eight out of eight restenotic lesions contained proliferating cells. The mean rate of proliferation was again higher in the restenotic (15.2 +/- 13.6%) than primary (3.6 +/- 3.5%) lesions (P < 0.05). Proliferating cells were detected as late as 1 yr after angioplasty. We conclude that cellular proliferation is a feature of atherosclerotic lesions which are associated with symptoms of ischemia, but that it is more prominent in restenosis compared to primary lesions. These findings have implications for therapies aimed at limiting lesion growth, particularly after percutaneous revascularization.

Antiproliferative gene BTG1 is highly expressed in apoptotic cells in macrophage-rich areas of advanced lesions in Watanabe heritable hyperlipidemic rabbit and human.

In the present study, the expression and potential role of the highly conserved antiproliferative gene BTG1 in the process of apoptosis as it occurs in atherosclerotic lesions was examined. In situ hybridization and immunodetection studies demonstrated that BTG1 localized to specific macrophage-rich regions of lesions in both Watanabe heritable hyperlipidemic rabbits and humans. In addition, the spatial distribution of BTG1 mRNA was shown to colocalize not only with macrophage-rich regions but also to cells that exhibited changes consistent with apoptosis, such as DNA fragmentation and nuclear condensation. In vitro studies demonstrated that forced overexpression of BTG1 induced a 3-fold increase in apoptosis in NIH/3T3 cells. Furthermore, significantly increased expression of BTG1 mRNA resulted from lipid loading of human monocyte-derived macrophages in vitro. The process of increasing lipid content in macrophages is frequently associated with decreased macrophage viability. Taken together, these data underscore a potentially important role for BTG1 in regulating the cellularity of advanced atherosclerotic lesions.

Effects of intracellular free cholesterol accumulation on macrophage viability: a model for foam cell death.

This study was designed to identify cellular responses associated with free cholesterol (FC) accumulation in model macrophage foam cells. Mouse peritoneal macrophages (MPMs) or J774 macrophages were loaded with cholesteryl esters using acetylated LDL and FC/phospholipid dispersions and were subsequently exposed to an acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor. This treatment produced a rapid accumulation of cellular FC. The FC that accumulated due to ACAT inhibition was more readily available for efflux to 2-hydroxypropyl-beta-cyclodextrin (which removes cholesterol from the plasma membrane) than FC in untreated control cells. After a 3-hour exposure to an ACAT inhibitor, a significant increase in phospholipid synthesis was seen, followed by the leakage of LDH after 12 hours of treatment. We also observed, by electron and fluorescence microscopy, morphological indications of both apoptosis and necrosis in cells treated with an ACAT inhibitor. In addition, inhibition of ACAT for 48 hours resulted in the formation of FC crystals in MPMs but not in J774 cells. If compound 3beta-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A), which modulates intracellular trafficking of cholesterol, was added together with the ACAT inhibitor, each of the metabolic changes elicited by the accumulation of excess FC was either diminished or eliminated. The protective affect of U18666A was not due to a decrease in cellular FC concentrations, because cells treated with an ACAT inhibitor accumulated similar amounts of FC in the presence or absence of U18666A. Thus, treatment with U18666A results in the sequestering of FC in a pool that prevents it from causing various responses to FC deposition in macrophages. The metabolic changes that were produced when these model foam cells were treated with the ACAT inhibitor parallel the pathological events that have been shown to occur in the developing atherosclerotic plaque.