Maternal Hypercholesterolemia During Pregnancy Promotes Early Atherogenesis in LDL Receptor-Deficient Mice and Alters Aortic Gene Expression Determined by Microarray

Abstract

Background-Maternal hypercholesterolemia during pregnancy is associated with markedly enhanced fatty streak formation in human fetal aortas and accelerated progression of atherosclerosis in normocholesterolemic children. Methods and Results-To establish the causal role of maternal hypercholesterolemia in a genetically homogeneous murine model and to test the hypothesis that pathogenic events during fetal development result in persistent changes in arterial gene expression, female LDL receptor-deficient (LDLR Ϫ/Ϫ ) mice were fed regular chow or high-fat diets supplemented with 0.075% or 1.25% cholesterol during pregnancy. Lesion sizes were determined in the aortic origin of their chow-fed offspring at 3 months. Maternal hypercholesterolemia more than doubled lesion sizes in male offspring (PϽ0.0001 for the 0.0075% cholesterol group). Microarray analysis of the expression of 11 000 murine genes in the nonatherosclerotic descending aorta by Affymetrix gene chips suggested that 139 genes were significantly regulated in offspring of hypercholesterolemic mothers. A subset of 12 of the upregulated transcripts was subjected to secondary analysis by semiquantitative PCR of pooled RNA and 4 genes were found to be upregulated Ͼ1.7-fold. Quantitative PCR for one of these genes using RNA from individual mice yielded similar results. Comparative immunostaining for several of the above genes also indicated increased protein content in offspring of hypercholesterolemic mothers. Conclusions-These findings establish an atherogenic effect of maternal hypercholesterolemia in genetically uniform mice and indicate that changes in aortic gene expression persist long after fetal exposure to hypercholesterolemia. In addition to elucidating pathogenic mechanisms initiated during fetal development, this approach may identify genes in morphologically normal arteries that influence the susceptibility to classical risk factors of atherosclerosis.

Long-term combined beneficial effects of physical training and metabolic treatment on atherosclerosis in hypercholesterolemic mice

“…The experiments conformed to the National Institutes of Health guidelines for the care of laboratory animals and were carried out on six groups of 8-week-old male low-density lipoprotein (LDL) receptor-deficient hypercholesterolemic mice (mean weight 42.4 Ϯ 13.2 g). We selected only male mice to avoid gender-related differences (49,50). The mice were randomized to one of three dietary interventions: (i) High-cholesterol diet alone (containing 21% by weight fat, 0.15% by weight cholesterol, and 19.5% by weight casein; Harlan Teklad, Madison, WI), as described (49)(50)(51); (ii) high-cholesterol diet plus antioxidant supplementation (1.0% vitamin E added to the chow and 0.05% vitamin C added to the drinking water); and (iii) high-cholesterol diet plus both antioxidants and L-arginine (6% in drinking water).…”

“…Mice were killed by CO 2 asphyxia, and then the blood was drawn into heparinized tubes from the inferior vena cava. The dissection of the aorta was performed under a stereomicroscope, as described in detail (46,(49)(50)(51). A 2-mm cross section was saved for immunohistochemical analysis, as described (46,(49)(50)(51).…”

“…The dissection of the aorta was performed under a stereomicroscope, as described in detail (46,(49)(50)(51). A 2-mm cross section was saved for immunohistochemical analysis, as described (46,(49)(50)(51). Tissue sections (5 m) were incubated with the E06 antibody (a generous gift of W. Palinski and J. L. Witztum, University of California at San Diego, La Jolla) against oxidation specific epitopes (46, 50), or with F4͞80, an antibody against mouse macrophage-derived foam cells (46,50).…”

“…Tissue sections (5 m) were incubated with the E06 antibody (a generous gift of W. Palinski and J. L. Witztum, University of California at San Diego, La Jolla) against oxidation specific epitopes (46, 50), or with F4͞80, an antibody against mouse macrophage-derived foam cells (46,50). The aorta was then cut longitudinally; one half was placed in 250 l of protein extraction buffer, and the other half was saved in 1 ml of KH 2 PO 4 buffer (46, 49-51) for further determination of eNOS protein expression (Western blot with rabbit polyclonal anti-human eNOS antibody) and the signal was detected by using chemiluminescence (Amersham Pharmacia ECL) as described (46,(49)(50)(51).…”

Apolipoprotein B Production Reduces Lipotoxic Cardiomyopathy

“…Reverse transcription used oligo(dT)24 with T7 promoter sequences attached followed by strand cDNA synthesis. cRNA was then amplified and biotinylated using T7 RNA polymerase (Enzo Diagnostics, Inc., Farmingdale, NY) prior to hybridization to the Affymetrix genechip mouse U74aV2 using the Affymetrix-recommended protocol (16). For comparison between experiments, expression data were globally scaled to an average intensity of 1000 using the Affymetrix Microarray Suite TM software.…”