Clusterin (14. Atherosclerosis )

Clusterin, a glycoprotein associated with subclasses of plasma high density lipoproteins (HDL), was found to accumulate in aortic lesions in a human subject with transplantation-associated arteriosclerosis and in mice fed a high-fat atherogenic diet. Foam cells present in mouse aortic valve lesions expressed clusterin mRNA, suggesting local synthesis contributes to clusterin‘s lo­calization in atherosclerotic plaque ^Ref .

Although clusterin was never observed in the normal aorta (ie, without any intimal lesions or intimal thickening), it was distributed not only in the intima but also in the media of aortas with diffuse, intimal thickening or atherosclerotic lesions. Double immunostaining with antibodies for clusterin and alpha-smooth muscle actin revealed clusterin deposition in smooth muscle cells (SMCs) or the aortic stroma in the vicinity of SMCs.the extent of clusterin distribution in the aortic wall increased with the degree of atherosclerosis development. In addition, the distribution pattern of clusterin was very similar to that of apoA-I and E. In situ hybridization with human clusterin cDNA demonstrated intense signals in cells scattered within the subendothelial space and medial SMCs of the aorta with advanced atherosclerosis but not in those of the normal aorta without intimal thickening. Furthermore, reverse transcriptase-polymerase chain reaction of the cultured human aortic SMCs revealed clusterin mRNA expression in these cells. The results indicate that clusterin in the aortic wall originates from not only clusterin circulated in the plasma but also clusterin produced by SMCs in the aortic wall. Considering the similarities of the distribution between clusterin and apo-A-I or E, it is hypothesized that clusterin possibly has a protective role against human therosclerosis by its involvement with cholesterol transport from the aortic wall to the liver ^Ref .

Mouse peritoneal macrophages were incubated with acetylated low-density lipoprotein (AcLDL) to produce foam cells containing cholesterol and cholesteryl esters. Incubation of the foam cells with physiological concentrations of purified clusterin led to a dose-dependent export of cholesterol. The appearance of cholesterol in the medium was associated predominantly with a decline in intracellular cholesteryl esters rather than intracellular free cholesterol. The kinetics of cholesterol release to clusterin were similar to apo A-I, an established promoter of cholesterol efflux. Clusterin was also shown to induce phospholipid efflux from cells, whereas the cholesterol exported to the medium was associated with clusterin. Studies using foam cells from apo E-null mice showed that the cholesterol exported to the medium was independent of apo E production by the cells. These results present the first evidence that clusterin can promote cholesterol efflux from foam cells and indicates that it might have a function in cellular cholesterol homoeostasis in both normal and pathological situations ^Ref .

Paraoxonase (Pon), clusterin, and apolipoprotein (apo) A-I accumulate in the artery wall during the development of atherosclerosis. In normal aortas (n = 6) there were low levels of extracellular Pon, clusterin, and apoA-I, immunoreactivity. The cytoplasm of smooth muscle cells in the media showed granular positivity for both Pon and apoA-I, indicating that these proteins were undergoing lysosomal degradation.this activity was also indicated by the presence of both intact and degradation products of Pon in smooth muscle cells as shown by Western blotting. With the progression of disease from fatty streaks (n = 3) to advanced atherosclerosis (n = 8) there was an increase in Pon, apoA-I, and clusterin immunoreactivity, indicating the increasing presence of these proteins with disease progression. These proteins are the components of a specific HDL subspecies that has been implicated in the prevention of peroxidative damage to phospholipids in LDL and membranes. The increase in Pon, clusterin, and apoA-I during the development of atherosclerosis may therefore represent a protective response to the oxidative stress associated with the development of atherosclerosis ^Ref .

The effects of mildly oxidized LDL and atherosclerosis on the levels of two proteins associated with HDL; clusterin, and paraoxonase (PON) were studied. On an atherogenic diet, PON activity decreased by 52%, and apoJ levels increased 2.8-fold in fatty streak susceptible mice, C57BL/6J (BL/6), but not in fatty streak resistant mice, C3H/HeJ (C3H). Plasma PON activity was also significantly decreased, and clusterin levels were markedly increased in apolipoprotein E knockout mice on the chow diet, resulting in a 9.2-fold increase in the clusterin/PON ratio as compared to controls. Furthermore, a dramatic increase in the clusterin/PON ratio (over 100-fold) was observed in LDL receptor knockout mice when they were fed a 0.15%-cholesterol-enriched diet. Injection of mildly oxidized LDL (but not native LDL) into BL/6 mice (but not in C3H mice) on a chow diet resulted in a 59% decrease in PON activity (P < 0.01) and a 3.6-fold increase in clusterin levels (P < 0.01). When an acute phase reaction was induced in rabbits, or the rabbits were placed on an atherogenic diet, hepatic mRNA for clusterin was increased by 2.7-fold and 2.8-fold, respectively. Treatment of HepG2 cells in culture with mildly oxidized LDL (but not native LDL) resulted in reduced mRNA levels for PON (3.0-fold decrease) and increased mRNA levels for clusterin (2.0-fold increase). In normolipidemic patients with angiographically documented coronary artery disease who did not have diabetes and were not on lipid-lowering medication (n = 14), the total cholesterol/HDL cholesterol ratio was 3.1+/-0.9 as compared to 2.9+/-0.4 in the controls (n = 19). This difference was not statistically significant. In contrast, the clusterin/PON ratio was 3.0+/-0.4 in the patients compared to 0.72+/-0.2 in the controls (P < 0.009). In a subset of these normolipidemic patients (n = 5), the PON activity was low (48+/-6.6 versus 98+/-17 U/ml for controls; P < 0.009), despite similar normal HDL levels, and the HDL from these patients failed to protect against LDL oxidation in co-cultures of human artery wall cells. It is concluded that: (a) mildly oxidized LDL can induce an increased clusterin/PON ratio, and (b) the clusterin/PON ratio may prove to be a better predictor of atherosclerosis than the total cholesterol/HDL cholesterol ratio ^Ref .

In a study analyzing the effects of the lecitin-chesterol acyltranferase gene knock-out, clusterin levels, rather than being decreased, were significantly (P = 0.01) higher (36%) in Lcat (-/-) than in Lcat (+/+) mice, and the clusterin/ PON ratio was 3-fold greater in Lcat (-/-) than in Lcat (+/+) animals112. A peptide was synthesized from D-amino acids corresponding to residues 113 to 122 in clusterin. D- [113–122]clusterin significantly improved the ability of plasma to promote cholesterol efflux and improved high-density lipoprotein (HDL) inflammatory properties for up to 48 hours after a single oral dose in apoE-null mice, whereas scrambled D- [113–122]clusterin did not. Oral administration of 125 microg/mouse/d of D- [113–122]clusterin reduced atherosclerosis in apoe-null mice (70.2% reduction in aortic root sinus lesion area, P=4.3×10(-13); 70.5% reduction by en face analysis, P=1.5×10(-6)). In monkeys, oral D- [113–122]clusterin rapidly reduced lipoprotein lipid hydroperoxides (LOOH) and improved HDL inflammatory properties. Adding 250 ng/ml of D-[113–122]clusterin (but not scrambled D- [113–122]clusterin) to plasma in vitro reduced LOOH and increased paraoxonase activity. Oral D- [113–122]clusterin significantly improves HDL inflammatory properties in mice and monkeys and inhibits lesion formation in apoe-null mice ^Ref .

An insertion (I)/deletion (D) polymorphism was found within the clusterin gene, D/D subjects had significantly higher levels of total cholesterol and low-density lipoprotein (LDL)-cholesterol than I/I subjects in females but not in males. Female subjects with the D allele (D/D+i/D) had greater intima-media thickness of the carotid artery than I/I subjects. In a multiple logistic regression analysis, the D allele of 6316delT was detected as an independent predictor for the plaque prevalence. In conclusion, the clusterin gene polymorphism may contribute to the serum lipid levels and the progression of carotid atherosclerosis in hypertensive Japanese females ^Ref .