Expression of Sonic Hedgehog downstream genes is modified in rat embryos exposed in utero to a distal inhibitor of cholesterol biosynthesis.

Holoprosencephaly is a common developmental anomaly of the forebrain and midface, that has been associated with mutations in the Sonic Hedgehog gene, and with perturbations of cholesterol synthesis and metabolism in mammalian embryos. The study presented here was aimed to evaluate the functional relationship between these two causal agents in the genesis of the phenotype. Therefore, we used rat embryos exposed in utero to a distal inhibitor of cholesterol biosynthesis (AY9944) in which we analyzed different Shh-dependent processes, as evaluated by the expression of eight target genes. In addition, to delineate between the impact of cholesterol shortage and/or sterol precursors accumulation on the Shh signaling cascade we exposed rat embryos to AY9944 and we provided complementary diets rich in cholesterol and 7-DHC. At the early-somite stage we observed a reduction of Shh signaling in AY9944 treated embryos, resulting in the definition of a narrower ventral domain. Later in development this reduction of Shh signaling led to a complete interruption of the pathway in the rostral hindbrain and caudal midbrain. Other regions such as the forebrain and the spinal cord appeared less sensitive to the reduction of Shh signaling and interruption of the pathway was only observed in a subset of embryos. Finally, we did provide evidence that 7-DHC accumulation is compatible with normal activity of Shh, as long as cholesterol levels in embryonic tissue is sufficient.

Role of cholesterol in embryonic development.

We showed previously that 3 distal inhibitors of cholesterol synthesis are highly teratogenic in rats. AY 9944 and BM 15766 inhibit 7-dehydrocholesterol reductase, which catalyzes the last step of cholesterol synthesis, and triparanol inhibits Delta(24)-dehydrocholesterol reductase, which catalyzes the last step in another pathway. These molecules cause holoprosencephalic brain anomalies. Under certain experimental conditions, other anomalies (of the limbs and male genitalia) are also observed. Assays performed by gas chromatography-mass spectrometry (GC-MS) show hypocholesterolemia and an accumulation of precursors. These data indicate that this animal model can be considered a model of Smith-Lemli-Opitz syndrome. Smith-Lemli-Opitz syndrome is a recessive autosomal genetic disease characterized by malformations (microcephaly, corpus callosum agenesis, holoprosencephaly, and mental retardation), male pseudohermaphroditism, finger anomalies, and failure to thrive. The syndrome has been attributed to a deficit in 7-dehydrocholesterol reductase. As assayed by GC-MS, the sterol status of these patients indicates severe hypocholesterolemia and an accumulation of precursors: 7-dehydrocholesterol, 8-dehydrocholesterol, and oxidized derivatives. The presence of 7-dehydrocholesterol in the serum of patients is pathognomonic of the disease. The developmental gene Shh (sonic hedgehog) plays a key role in brain, limb, and genital development; it was shown recently that the Shh protein has to be covalently linked to cholesterol to be active. This is the first time that a posttranslational function has been attributed to cholesterol. There is an obvious relation between Shh dysfunction and the malformations observed in our experiments and in patients with Smith-Lemli-Opitz syndrome. However, the exact relation remains to be clarified. It is clear, however, that the role of cholesterol in embryonic development must be taken into account.

Cholesterol deficit but not accumulation of aberrant sterols is the major cause of the teratogenic activity in the Smith-Lemli-Opitz syndrome animal model.

Low cholesterol and high 7-dehydrocholesterol (7DHC) levels are associated with a blockade of Delta7-reductase in the Smith-Lemli-Opitz syndrome (SLOS) and in the animals treated with the inhibitor AY9944. The impact of the cholesterol deficit and of the accumulation of 7DHC on the embryo were investigated in AY9944-treated pregnant rats receiving an enriched cholesterol or 7DHC diet. Sterol profiling was performed under the various nutritional conditions. AY9944 caused a severe decrease in the maternal and embryo cholesterol. The deficit in the embryo was sustained by the embryonic uptake of the inhibitor. A cholesterol-rich diet was efficient in restoring the maternal and embryonic cholesterol and phenotype but a 7DHC-rich diet did not modify the sterol status compared with dams treated with only AY9944. The offspring phenotype remained deleterious whether or not the dams received 7DHC-rich diet. Over 80% of the 7DHC was absorbed, as was cholesterol, which was not quantitatively influenced by AY9944. When cholesterol and 7DHC were simultaneously administered, a competition for intestinal absorption enhanced the lowering cholesterol effect of AY9944. Whether or not the dams received a 7DHC dietary supplement, the offspring's phenotype became normal when the diet was supplemented with cholesterol. Under conditions in which the ratio of cholesterol/7DHC is substantially varied, the normal development of embryos can be achieved as long as the cholesterol is sufficient. The phenotype is reversed in vivo by cholesterol which contrasts with the irreversible effects manifested in vitro by oxidized 7DHC by-products.

Oxidized derivatives of 7-dehydrocholesterol induce growth retardation in cultured rat embryos: a model for antenatal growth retardation in the Smith-Lemli-Opitz syndrome.

7-Dehydrocholesterol accumulates in fetuses affected by the Smith-Lemli-Opitz syndrome as a result of a deficit in the ultimate step of cholesterol synthesis catalyzed by Delta7 reductase. Rat embryos explanted at gestation day 10 and cultured for 48 h in the presence of the Delta7 reductase inhibitor AY 9944 were used as a model to discriminate between the beneficial effect of supplementation with cholesterol and the deleterious effect of supplementation with 7-dehydrocholesterol. Cholesterol supplementation in the form of mixed cholesterol/lecithin liposomes added to serum serving as the culture medium restores the growth of embryos which is markedly decreased in the presence of the inhibitor. 7-Dehydrocholesterol under identical conditions does not restore growth and impairs the beneficial effect of cholesterol added simultaneously. UV-photooxidation of 7-dehydrocholesterol-supplemented culture medium enhances its embryotoxicity, which suggests uptake by the embryo of toxic by-products formed from 7-dehydrocholesterol. By contrast photooxidation of cholesterol-supplemented culture medium does not induce embryotoxicity. alpha-Tocopherol reduces the toxicity of photooxidized 7-dehydrocholesterol supplementing the culture medium. We conclude that 7-dehydrocholesterol does not fulfill the cholesterol requirement of the developing embryos and exerts an additional embryotoxic effect probably via oxidized by-products. This could explain the antenatal growth retardation of SLOS by a blockage of the maternal compensatory cholesterol influx.

Inhibition of LPL expression in human monocyte-derived macrophages is dependent on LDL oxidation state: a key role for lysophosphatidylcholine.

The regulation of macrophage lipoprotein lipase (LPL) secretion and mRNA expression by atherogenic lipoproteins is of critical relevance to foam cell formation. LPL is present in arterial lesions and constitutes a bridging ligand between lipoproteins, proteoglycans, and cell receptors, thus favoring macrophage lipoprotein uptake and lipid accumulation. We investigated the effects of native and of oxidized lipoproteins on the expression of LPL in an in vitro human monocyte-macrophage system. Exposure of mature macrophages (day 12) to highly copper-oxidized human low density lipoprotein (LDL) (100 microg protein per milliliter) led to marked reduction in the expression of LPL activity (-62%, P<0.01) and mRNA level (-47%, P<0.05); native LDL, acetylated LDL, and LDL oxidized for <6 hours were without effect. The reduction in LPL activity became significant at a threshold of 6 hours of LDL oxidation (-31%, P<0.05). Among the biologically active sterols formed during LDL oxidation, only 7beta-hydroxycholesterol (5 microg/mL) induced a minor reduction in macrophage LPL activity, whereas 25-hydroxycholesterol was without effect. By contrast, lysophosphatidylcholine, whose LDL content increased in parallel with the degree of oxidation, induced significant reductions in LPL activity and mRNA levels at concentrations of 2 to 20 micromol/L (-34% to -53%, P<0.01). Our results demonstrate that highly oxidized LDL (>6-hour oxidation) exerts negative feedback on LPL secretion in human monocytes-macrophages via a reduction in mRNA levels. By contrast, native LDL and mildly oxidized LDL (<6-hour oxidation) did not exert a feedback effect on LPL expression. We speculate that the content of lysophosphatidylcholine and, to a lesser degree, of 7beta-hydroxycholesterol in oxidized LDLs is responsible for the downregulation of LPL activity and mRNA abundance in human monocyte-derived macrophages and may therefore modulate LPL-mediated pathways of lipoprotein uptake during conversion of macrophages to foam cells.