Mutant LRP6 Impairs Endothelial Cell Functions Associated with Familial Normolipidemic Coronary Artery Disease

Abstract

Mutations in the genes low-density lipoprotein (LDL) receptor-related protein-6 (LRP6) and myocyte enhancer factor 2A (MEF2A) were reported in families with coronary artery disease (CAD). We intend to determine the mutational spectrum of these genes among hyperlipidemic and normolipidemic CAD families. Forty probands with early-onset CAD were recruited from 19 hyperlipidemic and 21 normolipidemic Chinese families. We sequenced all exons and intron-exon boundaries of LRP6 and MEF2A, and found a novel heterozygous variant in LRP6 from a proband with normolipidemic CAD. This variant led to a substitution of histidine to tyrosine (Y418H) in an evolutionarily conserved domain YWTD in exon 6 and was not found in 1025 unrelated healthy individuals. Co-segregated with CAD in the affected family, LRP6Y418H significantly debilitated the Wnt3a-associated signaling pathway, suppressed endothelial cell proliferation and migration, and decreased anti-apoptotic ability. However, it exhibited no influences on low-density lipoprotein cholesterol uptake. Thus, mutation Y418H in LRP6 likely contributes to normolipidemic familial CAD via impairing endothelial cell functions and weakening the Wnt3a signaling pathway.

Keywords: LDL receptor-related protein-6 (LRP6); coronary artery disease; endothelial cell dysfunction; familial; normolipidemic.

Figure 1

Novel mutation of LRP6 identified in a CAD pedigree. (A) Pedigree of the family with the LRP6 Y418H mutation. Numbered individuals correspond to those in Table 1. Circles represent females; Squares represent males; Proband is indicated by the arrow; Individuals with CAD are indicated by black symbols; Individuals without CAD are shown as unfilled symbols; Presymptomatic carriers are shown by symbols by gray symbols; Symbols with a slash through them indicate deceased subjects; Genotypes of the LRP6 mutation were shown below the symbols who were willing to participate in the study; Filled stars indicates that the genotype of subject I2 was speculated; Individuals who were not available for studied are indicated with question mark; (B) Coronary angiogram of the proband. The red arrows indicates the stenosis; (C) DNA sequence analysis for a segment of LRP6 exon 6 from a healthy control (top) and the proband (below). A red arrow points out a single base mutation in the proband, and it results in the substitution of histidine for tyrosine at codon 418; (D) Conservatism analysis by interspecies alignments. The mutation position is indicated with a red frame.

Figure 2

Effect of LRP6_Y418H and LRP6_R611C on Wnt signal transduction. (A) RT-PCR showed that there was no significant difference between the over-expression levels of LRP6_WT/LRP6_R611C/LRP6_Y418H; (B) Western blot showed no significant difference between total expression levels or membrane location of wild-type and mutant LRP6. Results were replicated three times and a representative figure was shown; (C,D) Statistical result of (B); (E) Luciferase assay was performed with different amount of Wnt3a. RLU: relative light units. Results were obtained with four independent transfections. Error bars, standard deviation. * indicate p-value for one-way ANOVA plus post-hoc test <0.05. NS, not significant in one-way ANOVA plus post-hoc test.

Figure 3

Effect of LRP6_Y418H and LRP6_R611C on endothelial cell functions; (A) Comparison of endothelial cells’ proliferation. The same amount of cells was over-expressed with wild-type LRP6 or mutant. After 48 h, cell number was counted for each group. Results were calculated using eight random fields from four independent biological replications; (B) Comparison of endothelial cell migration. Results were calculated using six views from three independent replications of Boyden chamber assay; (C) Electrophorogram for DNA ladder assay. SD, serum depletion; (D) Scatter diagram from flow cytometry assay. Data were presented in 2D diagrams plotting PI against Annexin-APC. Compensation for background fluorescence was performed by measuring target signals of single color controls and negative controls. Two quadrants in the right-side diagram represent apoptotic cells; (E) Statistical result for proportion of apoptotic cells in each group. Three biological repeats were taken into calculation; (F–J) Relative mRNA level of markers for endothelial cell activation (IL6, SELE, and ICAM) and senescence (P21 and P16). Three biological repeats were taken into calculation. Error bars, standard deviation. * indicate p-value for one-way ANOVA plus post-hoc test <0.05. NS, not significant.

Figure 4

Effect of LRP6_Y418H and LRP6_R611C on LDL uptake in HUVEC. (A) An overview of the Dil-LDL uptake in HUVEC; Left, bright field; Right, Fluorescent field; (B) Quantitative results were calculated using six random views from three independent replications. Error bars, standard deviation. * indicate p-value for one-way ANOVA plus post-hoc test <0.05. NS, not significant; (C) Enlarged views for single cells. Top, GFP translated by IRES following LRP6; Middle, Dil-LDL; Bottom, merged data.