Mari Niihashi

Fluvastatin suppresses atherosclerotic progression, mediated through its inhibitory effect on endothelial dysfunction, lipid peroxidation, and macrophage deposition

Fluvastatin, a potent 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, exerts an inhibitory effect on intimal thickening after mechanical injury in normocholesterolemic rabbit artery at a dose not enough to elicit a known action of lipid lowering. This study was designed to determine whether atherosclerotic progression triggered by hypercholesterolemia can be inhibited by fluvastatin under conditions without its hypocholesterolemic effect. Rabbits were fed a 0.5% cholesterol diet or normal diet for 17 weeks and were treated with either fluvastatin (0.3-2 mg/kg/day, p.o.) or pravastatin (2 mg/kg/day, p.o.). Atherogenic features manifested in the cholesterol-diet group, compared with the normal-diet group; they were the increase in serum lipid peroxide level, in the intraluminal lesion area of the aorta, and in macrophage content of the aortic cross-sectional lesion area; the attenuation of endothelium-dependent relaxing response to acetylcholine in the femoral artery; and the increase in serum lipid level. Treatment with fluvastatin, but not pravastatin, inhibited the manifestation of the atherogenic features without a serum lipid-lowering effect. Thus fluvastatin is likely to reduce the risk of atherosclerotic progression, to which endothelial dysfunction, lipid peroxidation, and macrophage accumulation in the vasculature may contribute, irrespective of changes in serum lipid levels.

Inhibitory effect of fluvastatin at doses insufficient to lower serum lipids on the catheter-induced thickening of intima in rabbit femoral artery.

The anti-atherosclerotic effect of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors at doses insufficient to lower serum cholesterol was investigated in rabbit femoral artery denuded by balloon catheter. Fluvastatin and pravastatin were given orally at doses of 4 and 8 mg/kg per day, respectively, for 2 weeks after the catheterization. There was little change in serum cholesterol, triglyceride and phospholipid by chronic treatment with the drugs. The cross-sectional area of the intima, expressed as relative values to media (I/M ratio), was increased by the catheterization, showing intimal thickening in the denuded arteries. The I/M ratio was reduced by fluvastatin but not pravastatin: 0.327 +/- 0.060 for control, 0.116 +/- 0.035 for 4 mg/kg fluvastatin, 0.088 +/- 0.027 for 8 mg/kg fluvastatin and 0.22 +/- 0.069 for 8 mg/kg pravastatin. Fluvastatin (8 mg/kg)-induced effect on the I/M ratio, was prevented by the combined administration with 40 mg/kg per day mevalonate, a metabolite in the HMG-CoA reductase pathway. These results suggest that fluvastatin inhibits intimal thickening after catheterization-induced injury through percutaneous transluminal coronary angioplasty (PTCA) and that the inhibition is presumably attributed to reduced migration and proliferation of smooth muscle cells but not secondarily to a lowering of serum lipid.

Increased expression of peroxisome proliferator-activated receptor (PPAR)-α and PPAR-γ in human atherosclerosis

To clarify the mechanism of atherosclerosis development in humans, we studied the mRNA and protein expression of PPAR subtypes in various types of atherosclerotic lesions and their correlation with cell proliferation and macrophage invasion. Human aortas were obtained from 35 autopsied cases, and each sample was divided into halves. One half was used for the analysis of mRNA or protein expression with RT-PCR or Western blotting, respectively. The other was microscopically classified into atheromatous plaque (AP), fatty streak (FS), and diffuse intimal thickening (DIT), and was analyzed immunohistochemically. The mRNA levels of both PPARs increased significantly in atherosclerosis and tended to increase in proportion to the severity of the lesion, and the expression of PPAR-alpha correlated with that of PPAR-gamma in FS and AP. The PPAR-gamma protein increased in AP. Monocytes/macrophages, as well as endothelial and smooth muscle cells, expressed the PPAR-gamma protein in plaques. This expression in the DIT was noted mainly in macrophages but was not correlated with the density of macrophages, suggesting that only certain macrophages express the PPARs in DIT. Cell proliferation did not correlate with PPARs expression in any lesion type. These findings suggest that PPARs may be associated with atheromatous plaque formation, and that PPAR-gamma may be involved in the early stages of human atherosclerosis.

Expression of Muscarinic Receptor Genes in the Human Coronary Artery

The authors investigated the role of muscarinic receptors in functional control of coronary arteries affected by intimal thickening due to arteriosclerosis. They first examined the genetic subtypes of muscarinic receptors expressed in human coronary arteries. Twelve samples of human coronary artery, obtained by autopsy from eight subjects, were examined for the expression of four genetic subtypes of muscarinic receptor, m1 to m4, by reverse transcription and polymerase chain reaction (RT-PCR). Two subtypes, m2 and m3, were found to be expressed in the coronary artery. The m2 gene was expressed in seven of the 12 vessels, and m3 in eight of the 12. Expression of both m2 and m3 genes was observed in five of the 12 vessels. Neither the m1 nor m4 was expressed in these samples. These results indicate that the m2 and m3 genes are mainly expressed in the coronary arteries and suggest that these patterns of expression are differentially controlled to induce the diversity of contraction/relaxation reactions induced in the coronary arteries by acetylcholine.

Correlation of two argyrophilic nucleolar organizer region counting methods with the Ki-67 labeling index in uterine smooth muscle cell tumors

The argyrophilic nucleolar organizer regions (AgNOR) are silver stained granules that are thought to correlate with cell proliferation activity. Two AgNOR counting methods: the mean AgNOR count (mAgNOR, the mean number of AgNOR granules in 100 cells) and the AgNOR proliferatlve Index (pAgNOR, the percentage of cells exhibiting five or more AgNOR granules per nuclei) have been proposed. In this study, the two counting methods were applied to 58 cases of normal uterine corpus and uterine corpus tumors and were compared with the Ki‐67 labeling Index (Ki‐67 Ll) using MIB‐1 monoclonal antibody and other hlstopathologlcal criteria. Notable differences In the number of AgNOR and the Ki‐67 Ll were observed between benign and malignant smooth muscie tissue. Hlstopathologic features are well correlated to the proliferatlve activity of tumors. Although the most reliable method of predicting malignant potential cannot be determined, the methods outlined by this study are thought to be highly useful In assessing proliferatlve activities.

Three‐dimensional Reorganization of a Cell Line of Papilla Vateri Adenocarcinoma in Various Culture Conditions

SAV‐I, a cell line derived from a well differentiated adenocarcinoma of Vaters papilla, was cultured under four different conditions using collagen gel matrices (type I collagen): 1) double‐layered, 2) floating double‐layered, 3) embedded, and 4) floating embedded, then observed by light and electron microscopy and immunohistochemistry. Under all four conditions, three‐dimensional growth with tubules occurred. In particular, the floating double‐layered condition, where the cells were cultured between two collagen gel layers, then floated onto the medium, was useful for showing cellular reorganization. The three‐dimensional growth patterns observed in vitro closely resembled the in vivo growth of SAV I cells transplanted into nude mice. Therefore, we conclude that the floating double‐layered condition is useful for demonstrating the morphological characteristics of the parent cells of established cell lines, and should be advantageous for studies of the relationship between cellular morphology and function in vitro.