LTP is impaired in <i>Lrp4</i>^{<i>ECD/ECD</i>} but not in <i>Lrp4</i>^{<i>ΔICD/ΔICD</i>} mice.

<p><b>A</b>: <i>Upper Panel</i>, Sample traces before and 40 min after theta-burst stimulation (TBS); <i>Lower Panel</i>, Results of experiments from <i>Lrp4</i>^{<i>ECD/ECD</i>} mice compared to their <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>} controls. TBS induced on average a 37.63 ± 7.88% increase in <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>} control slices (open squares, n = 16, N = 5), but only 14.83 ± 3.39% LTP in slices from the <i>Lrp4</i>^{<i>ECD/ECD</i>} mice (black triangles, n = 10, N = 3). <b>B</b>: Unpaired t-test was used to compare each sample for LTP calculated 40–60 min after theta-burst. Values are the means of the normalized fEPSP slopes. * denotes significance, p = 0.0387. <b>C:</b><i>Upper Panel</i>, Sample traces before and 40 min after TBS. <i>Lower Panel</i>, Results of experiments from <i>Lrp4</i>^{<i>ΔICD/ΔICD</i>} mice compared to their <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>} controls. <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>} slices were recorded on consecutive days and used as internal controls and pooled together. TBS induced a 37.63 ± 7.88% LTP in <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>} control slices (open squares, n = 16, N = 5), and 32.42 ± 6.27% LTP in slices from the <i>Lrp4</i>^{<i>ΔICD/ΔICD</i>} mice (gray filled rhombus, n = 11, N = 5). <b>D</b>: There was no significant difference in LTP between <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>}<i>and Lrp4</i>^{<i>ΔICD/ΔICD</i>} mice (p = 0.63). <b>E.</b> Input-output curves calculated as a function of fiber volley amplitude to the slopes of fEPSP’s. Average peak amplitudes for <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>}, <i>Lrp4</i>^{<i>ECD/ECD</i>} and <i>Lrp4</i>^{<i>ΔICD/ΔICD</i>} slices used in the experiments were 1.68 ± 0.15 mV, 1.27 ± 0.25 mV, and 1.50 ± 0.20 mV), respectively, and were not significantly different from each other. (One-way ANOVA, F = 1.124, p = 0.33.) <b>F:</b> Theta-burst analysis or <b>G:</b> paired pulse ratios (n = 8, N = 3 for each) did not reveal any significant differences between <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>} and <i>Lrp4</i>^{<i>ECD/ECD</i>} (two-way ANOVA, F(3,56) = 0.46, p = 0.71). N = number of animals.</p

Normal brain development in <i>Lrp4</i>^{<i>ECD/ECD</i>} and <i>Lrp4</i>^<i>-/-</i> mice.

<p><b>A-D</b>: Sagittal slices of the <i>Lrp4</i>^{<i>ECD/ECD</i>} (A,C) and wild type (<i>Lrp4</i>^<i>+/+</i> B,D) mouse cerebellum labeled with NeuN (green), Brn1 (red) and DAPI (blue). Brn1 and NeuN are commonly used markers to label neurons. ML = molecular layer, PL = Purkinje cell layer, and GCL = granule cell layer are clearly distinguishable and not different in the cerebellum of <i>Lrp4</i>^{<i>ECD/ECD</i>} and <i>Lrp4</i>^<i>+/+</i> adult mice (>2 months). <b>E-H</b>: Coronal sections of <i>Lrp4</i>^{<i>ECD/ECD</i>} (E,F) and <i>Lrp4</i>^<i>+/+</i> (G,H) brains showing hippocampus (E,G) and somatosensory cortex (F,H). Slices are labeled for NeuN and DAPI to visualize normal cortical lamination (layers I-VI). I-N: Coronal sections of E18.5 <i>Lrp4</i>^<i>-/-</i> brains compared to their wild type litter mates. Brn1 (I,J) and GFAP (K,L) immunoreactivity in the cortex and hippocampus and Tbr1 plus NeuN double labeling in the cortex are illustrated. Scale bars = 200 μm (A-H), 400 μm (I-L), 100 μm (M,N).</p

Limb and bone structure of different <i>Lrp4</i> KI mutants.

<p><b>A</b>: Illustration of the different Lrp4 protein products of all KI mutants. Panels are aligned to paw images in B and C to indicate genotypes. <b>B</b>: Ventral view of fore and hind limbs of <i>Lrp4</i> KI mutants. Homozygous mutant mice for each allelic variant (<i>KI/KI</i>) and compound mutant mice that carry one allelic variant and one KO allele (<i>KO/KI</i>) are shown. Note that there are strong defects in the limb pattering of <i>Lrp4</i>^{<i>ECD/ECD</i>}, intermediate defects in <i>Lrp4</i>^{<i>ΔICD/ΔIC</i>}, and only mild defects in <i>Lrp4</i>^{<i>LDLR-ICD/LDLR-ICD</i>} (red arrows). <b>C</b>: Ventral view of alizarin red (stains bones) and alcian blue (stains cartilage) of different <i>Lrp4</i> KI mutants. A WT-KI allele (2^nd panel in A and B) was generated to control for the lack of introns in the ICD-cassette in the other KI mutants. Black arrowheads: ectopic bone or bony fusion; red arrowheads: soft-tissue fusion. (modified from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116701#pone.0116701.ref022" target="_blank">22</a>]).</p

Data_Sheet_1_Dietary-Induced Elevations of Triglyceride-Rich Lipoproteins Promote Atherosclerosis in the Low-Density Lipoprotein Receptor Knockout Syrian Golden Hamster.docx

Elevated triglycerides are associated with an increased risk of cardiovascular disease (CVD). Therefore, it is very important to understand the metabolism of triglyceride-rich lipoproteins (TRLs) and their atherogenic role in animal models. Using low-density lipoprotein receptor knockout (LDLR−/−) Syrian golden hamsters, this study showed that unlike LDLR−/− mice, when LDLR−/− hamsters were fed a high cholesterol high-fat diet (HFD), they had very high plasma levels of triglycerides and cholesterol. We found that LDLR−/− hamsters exhibited increased serum TRLs and the ApoB100 and 48 in these particles after being fed with HFD. Treatment with ezetimibe for 2 weeks decreased these large particles but not the LDL. In addition, ezetimibe simultaneously reduced ApoB48 and ApoE in plasma and TRLs. The expression of LRP1 did not change in the liver. These findings suggested that the significantly reduced large particles were mainly chylomicron remnants, and further, the remnants were mainly cleared by the LDL receptor in hamsters. After 40 days on an HFD, LDLR−/− hamsters had accelerated aortic atherosclerosis, accompanied by severe fatty liver, and ezetimibe treatment reduced the consequences of hyperlipidemia. Compared with the serum from LDLR−/− hamsters, that from ezetimibe-treated LDLR−/− hamsters decreased the expression of vascular adhesion factors in vascular endothelial cells and lipid uptake by macrophages. Our results suggested that in the LDLR−/− hamster model, intestinally-derived lipoprotein remnants are highly atherogenic and the inflammatory response of the endothelium and foam cells from macrophages triggered atherosclerosis. The LDL receptor might be very important for chylomicrons remnant clearance in the Syrian golden hamster, and this may not be compensated by another pathway. We suggest that the LDLR−/− hamster is a good model for the study of TRLs-related diseases as it mimics more complex hyperlipidemia.</p

DataSheet_1_CRISPR/Cas9 based blockade of IL-10 signaling impairs lipid and tissue homeostasis to accelerate atherosclerosis.pdf

Interleukin-10 (IL-10) is a widely recognized immunosuppressive factor. Although the concept that IL-10 executes an anti-inflammatory role is accepted, the relationship between IL-10 and atherosclerosis is still unclear, thus limiting the application of IL-10-based therapies for this disease. Emerging evidence suggests that IL-10 also plays a key role in energy metabolism and regulation of gut microbiota; however, whether IL-10 can affect atherosclerotic lesion development by integrating lipid and tissue homeostasis has not been investigated. In the present study, we developed a human-like hamster model deficient in IL-10 using CRISPR/Cas9 technology. Our results showed that loss of IL-10 changed the gut microbiota in hamsters on chow diet, leading to an increase in lipopolysaccharide (LPS) production and elevated concentration of LPS in plasma. These changes were associated with systemic inflammation, lipodystrophy, and dyslipidemia. Upon high cholesterol/high fat diet feeding, IL-10-deficient hamsters exhibited abnormal distribution of triglyceride and cholesterol in lipoprotein particles, impaired lipid transport in macrophages and aggravated atherosclerosis. These findings show that silencing IL-10 signaling in hamsters promotes atherosclerosis by affecting lipid and tissue homeostasis through a gut microbiota/adipose tissue/liver axis.</p

Data_Sheet_1_Targeting ApoC3 Paradoxically Aggravates Atherosclerosis in Hamsters With Severe Refractory Hypercholesterolemia.docx

RationaleApoC3 plays a central role in the hydrolysis process of triglyceride (TG)-rich lipoproteins mediated by lipoprotein lipase (LPL), which levels are positively associated with the incidence of cardiovascular disease (CVD). Although targeting ApoC3 by antisense oligonucleotide (ASO), Volanesorsen markedly reduces plasma TG level and increase high-density lipoprotein cholesterol (HDL-C) in patients with hypertriglyceridemia (HTG), the cholesterol-lowering effect of ApoC3 inhibition and then the consequential outcome of atherosclerotic cardiovascular disease (ASCVD) have not been reported in patients of familial hypercholesterolemia (FH) with severe refractory hypercholesterolemia yet.ObjectiveTo investigate the precise effects of depleting ApoC3 on refractory hypercholesterolemia and atherosclerosis, we crossed ApoC3-deficient hamsters with a background of LDLR deficiency to generate a double knockout (DKO) hamster model (LDLR−/−, XApoC3−/−, DKO).Approach and ResultsOn the standard laboratory diet, DKO hamsters had reduced levels of plasma TG and total cholesterol (TC) relative to LDLR−/− hamsters. However, upon high-cholesterol/high-fat (HCHF) diet feeding for 12 weeks, ApoC3 deficiency reduced TG level only in female animals without affecting refractory cholesterol in the circulation, whereas apolipoprotein A1 (ApoA1) levels were significantly increased in DKO hamsters with both genders. Unexpectedly, loss of ApoC3 paradoxically accelerated diet-induced atherosclerotic development in female and male LDLR−/− hamsters but ameliorated fatty liver in female animals. Further analysis of blood biological parameters revealed that lacking ApoC3 resulted in abnormal platelet (PLT) indices, which could potentially contribute to atherosclerosis in LDLR−/− hamsters.ConclusionsIn this study, our novel findings provide new insight into the application of ApoC3 inhibition for severe refractory hypercholesterolemia and ASCVD.</p