J.A. Berti

Atherosclerosis is enhanced by testosterone deficiency and attenuated by CETP expression in transgenic mice

In this work, we investigated the impact of testosterone deficiency and cholesteryl ester transfer protein (CETP) expression on lipoprotein metabolism and diet-induced atherosclerosis. CETP transgenic mice and nontransgenic (nTg) littermates were studied 4 weeks after bilateral orchidectomy or sham operation. Castrated mice had an increase in the LDL fraction (+36% for CETP and +79% for nTg mice), whereas the HDL fraction was reduced (−30% for CETP and −11% for nTg mice). Castrated mice presented 1.7-fold higher titers of anti-oxidized LDL (Ox-LDL) antibodies than sham-operated controls. Plasma levels of CETP, lipoprotein lipase, and hepatic lipase were not changed by castration. Kinetic studies showed no differences in VLDL secretion rate, VLDL-LDL conversion rate, or number of LDL and HDL receptors. Competition experiments showed lower affinity of LDL from castrated mice for tissue receptors. Diet-induced atherosclerosis studies showed that testosterone deficiency increased by 100%, and CETP expression reduced by 44%, the size of aortic lesion area in castrated mice. In summary, testosterone deficiency increased plasma levels of apolipoprotein B-containing lipoproteins (apoB-LPs) and anti-OxLDL antibodies, decreased LDL receptor affinity, and doubled the size of diet-induced atherosclerotic lesions. The expression of CETP led to a milder increase of apoB-LPs and reduced atherosclerotic lesion size in testosterone-deficient mice.

Cholesteryl ester transfer protein (CETP) increases postprandial triglyceridaemia and delays triacylglycerol plasma clearance in transgenic mice.

The CETP (cholesteryl ester transfer protein) is a plasma protein synthesized in several tissues, mainly in the liver; CETP reduces plasma HDL (high-density lipoprotein) cholesterol and increases the risk of atherosclerosis. The effect of CETP levels on postprandial intravascular metabolism of TAGs (triacylglycerols) is an often-overlooked aspect of the relationship between CETP and lipoprotein metabolism. Here, we tested the hypothesis that CETP delays the plasma clearance of TAG-rich lipoprotein by comparing human CETP expressing Tg (transgenic) and non-Tg mice. After an oral fat load, the postprandial triglyceridaemia curve was markedly increased in CETP-Tg compared with non-Tg mice (280+/-30 versus 190+/-20 mg/dl per 6 h respectively, P<0.02). No differences in intestinal fat absorption and VLDL (very-low-density lipoprotein) secretion rates were observed. Kinetic studies of double-labelled chylomicron-like EMs (emulsions) showed that both [(3)H]triolein and [(14)C]cholesteryl oleate FCRs (fractional clearance rates) were significantly reduced ( approximately 20%) in CETP-Tg mice. Furthermore, TAG from lipid EM pre-incubated with CETP-Tg plasma had plasma clearance and liver uptake significantly lower than the non-Tg plasma-treated lipid EM. In addition, reductions in post-heparin plasma LPL (lipoprotein lipase) activity (50%) and adipose tissue mRNA abundance (39%) were verified in CETP-Tg mice. Therefore we conclude that CETP expression in Tg mice delays plasma clearance and liver uptake of TAG-rich lipoproteins by two mechanisms: (i) transferring TAG to HDLs and increasing CE content of the remnant particles and (ii) by diminishing LPL expression. These findings show that the level of CETP expression can influence the responsiveness to dietary fat and may lead to fat intolerance.

Ciprofibrate increases cholesteryl ester transfer protein gene expression and the indirect reverse cholesterol transport to the liver

BackgroundCETP is a plasma protein that modulates atherosclerosis risk through its HDL-cholesterol reducing action. The aim of this work was to examine the effect of the PPARα agonist, ciprofibrate, on the CETP gene expression, in the presence and absence of apolipoprotein (apo) CIII induced hypertriglyceridemia, and its impact on the HDL metabolism.ResultsMice expressing apo CIII and/or CETP and non-transgenic littermates (CIII, CIII/CETP, CETP, non-Tg) were treated with ciprofibrate during 3 weeks. Drug treatment reduced plasma triglycerides (30-43%) and non-esterified fatty acids (19-47%) levels. Cholesterol (chol) distribution in plasma lipoprotein responses to ciprofibrate treatment was dependent on the genotypes. Treated CIII expressing mice presented elevation in VLDL-chol and reduction in HDL-chol. Treated CETP expressing mice responded with reduction in LDL-chol whereas in non-Tg mice the LDL-chol increased. In addition, ciprofibrate increased plasma post heparin lipoprotein lipase activity (1.3-2.1 fold) in all groups but hepatic lipase activity decreased in treated CETP and non-Tg mice. Plasma CETP activity and liver CETP mRNA levels were significantly increased in treated CIII/CETP and CETP mice (30-100%). Kinetic studies with 3H-cholesteryl ether (CEt) labelled HDL showed a 50% reduction in the 3H-CEt found in the LDL fraction in ciprofibrate treated compared to non-treated CETP mice. This means that 3H-CEt transferred from HDL to LDL was more efficiently removed from the plasma in the fibrate treated mice. Accordingly, the amount of 3H-CEt recovered in the liver 6 hours after HDL injection was increased by 35%.ConclusionTogether these data showed that the PPARα agonist ciprofibrate stimulates CETP gene expression and changes the cholesterol flow through the reverse cholesterol transport, increasing plasma cholesterol removal through LDL.

Effects of diabetes and CETP expression on diet-induced atherosclerosis in LDL receptor-deficient mice.

The role of CETP expression and diabetes in atherogenesis was investigated in mice with heterozygous disruption of the LDL receptor gene (LDLR1). LDLR1 mice with and without CETP expression were treated with streptozotocin (STZ) and maintained on a standard diet for one month before switching to an atherogenic diet for an additional month. STZ‐sensitive mice had ∼2.5‐fold higher glycemia and 7.5‐ to 8.0‐fold higher cholesterolemia. Factorial analysis of variance showed no significant effect of diabetes, CETP or diabetes‐CETP interaction on the size of the atherosclerotic lesions. CETP expression in non‐diabetic mice resulted in a 50% reduction in the area of the atherosclerotic lesions. Multiple regression analysis showed a positive and independent atherogenic effect of triglyceridemia in LDLR1 mice and of cholesterolemia in diabetic mice. Logistic analysis showed that elevated plasma cholesterol level significantly increased the risk of developing large lesion size (>75th percentile). In conclusion, CETP expression did not alter the lesion formation in response to diabetes, although it may be protective in the euglycemic state; the triglyceride level was an independent risk factor for LDL receptor‐deficient mice but not for CETP‐expressing mice; and elevated plasma cholesterol levels increased the risk of developing large atherosclerotic lesions, independently of CETP and diabetes.

Atherosclerosis in aged mice over-expressing the reverse cholesterol transport genes

We determined whether over-expression of one of the three genes involved in reverse cholesterol transport, apolipoprotein (apo) AI, lecithin-cholesterol acyl transferase (LCAT) and cholesteryl ester transfer protein (CETP), or of their combinations influenced the development of diet-induced atherosclerosis. Eight genotypic groups of mice were studied (AI, LCAT, CETP, LCAT/AI, CETP/AI, LCAT/CETP, LCAT/AI/CETP, and non-transgenic) after four months on an atherogenic diet. The extent of atherosclerosis was assessed by morphometric analysis of lipid-stained areas in the aortic roots. The relative influence (R2) of genotype, sex, total cholesterol, and its main sub-fraction levels on atherosclerotic lesion size was determined by multiple linear regression analysis. Whereas apo AI (R2 = 0.22, P < 0.001) and CETP (R2 = 0.13, P < 0.01) expression reduced lesion size, the LCAT (R2 = 0.16, P < 0.005) and LCAT/AI (R2 = 0.13, P < 0.003) genotypes had the opposite effect. Logistic regression analysis revealed that the risk of developing atherosclerotic lesions greater than the 50th percentile was 4.3-fold lower for the apo AI transgenic mice than for non-transgenic mice, and was 3.0-fold lower for male than for female mice. These results show that apo AI overexpression decreased the risk of developing large atherosclerotic lesions but was not sufficient to reduce the atherogenic effect of LCAT when both transgenes were co-expressed. On the other hand, CETP expression was sufficient to eliminate the deleterious effect of LCAT and LCAT/AI overexpression. Therefore, increasing each step of the reverse cholesterol transport per se does not necessarily imply protection against atherosclerosis while CETP expression can change specific atherogenic scenarios.

Soy protein containing isoflavones favorably influences macrophage lipoprotein metabolism but not the development of atherosclerosis in CETP transgenic mice

The possibility that soy protein containing isoflavones influences the development of experimental atherosclerosis has been investigated in ovariectomized mice heterozygous for the human CETP transgene and for the LDL-receptor null allele (LDLr+/− CETP+/−). After ovariectomy at 8 wk of age they were fed a fat/cholesterol-rich diet for 19 wk and divided into three experimental groups: dietary unmodified soy protein containing isoflavones (mg/g of diet), either at low-dose (Iso Low, 0.272, n=25), or at high-dose (Iso High, 0.535, n=28); and the atherogenic diet containing an isoflavone-depleted alcohol-washed soy protein as a control group (n=28). Aortic root lipid-stained lesion area (mean μm2×103±SD) did not differ among Iso Low (12.3±9.9), Iso High (7.4±6.4), and controls (10.7±12.8). Autoantibody titers against plasma oxidized LDL did not differ among the experimental groups. Using the control mice as the reference value (100%), in vitro mouse peritoneal macrophage uptake of labeled acetylated LDL-cholesterol was lower in the Iso High (68%) than in the Iso Low (85%) group. The in vitro percent removal by exogenous HDL of labeled unesterified cholesterol from macrophages previously enriched with human [4-14C]-cholesteryl oleate acetylated LDL was enhanced in the Iso High group (50%). In spite of these in vitro potentially antiatherogenic actions, soy protein containing isoflavones did not modify the average size of lipid-stained area in the aortic root.