Deficiency of ASGR1 in pigs recapitulates reduced risk factor for cardiovascular disease in humans

Abstract

Genetic variants in the asialoglycoprotein receptor 1 (ASGR1) are associated with a reduced risk of cardiovascular disease (CVD) in humans. However, the underlying molecular mechanism remains elusive. Given the cardiovascular similarities between pigs and humans, we generated ASGR1-deficient pigs using the CRISPR/Cas9 system. These pigs show age-dependent low levels of non-HDL-C under standard diet. When received an atherogenic diet for 6 months, ASGR1-deficient pigs show lower levels of non-HDL-C and less atherosclerotic lesions than that of controls. Furthermore, by analysis of hepatic transcriptome and in vivo cholesterol metabolism, we show that ASGR1 deficiency reduces hepatic de novo cholesterol synthesis by downregulating 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), and increases cholesterol clearance by upregulating the hepatic low-density lipoprotein receptor (LDLR), which together contribute to the low levels of non-HDL-C. Despite the cardioprotective effect, we unexpectedly observed mild to moderate hepatic injury in ASGR1-deficient pigs, which has not been documented in humans with ASGR1 variants. Thus, targeting ASGR1 might be an effective strategy to reduce hypercholesterolemia and atherosclerosis, whereas further clinical evidence is required to assess its hepatic impact.

Fig 1. Generation of ASGR1 knockout pigs.

A. Schematic diagram of Cas9-sgRNA targeting sites of the pig ASGR1 locus. Two sgRNAs were used to target pig ASGR1 exon 6 to promote DNA breaks and homologous recombination. The sgRNA targeting sequences are shown in red, and the PAM sequences are shown in blue and are underlined. E1-E9: Exon 1–9; the black arrow: Site of the start codon; the red arrow: primer site of the genotyping. B. Western blots of ASGR1 and internal control β-actin in the livers of 24-month-old ASGR1^-/- pigs (the -20 bp / -20 bp and the -137 bp / +1 bp genotypes, n = 3 in each phenotype) and age-matched WT pigs (n = 6). C. Representative photograph of founder (ASGR1^-/-) pigs and F1 (ASGR1^+/-) pigs. PAM, protospacer adjacent motif; WT, wild-type.

Fig 2. Plasma lipoprotein profiles of pigs under standard diet.

A-D. The effect of age on levels of non-HDL-C, LDL-C, HDL-C, TC, VLDL-C TG, ApoA1 and ApoB in ASGR1^-/- pigs and WT controls receiving a standard diet (n = 6 for ASGR1^-/- pigs, n = 10 for WT controls. Data represent two independent experiments combined). E-H. The effect of age on levels of non-HDL-C, LDL-C, HDL-C, TC, VLDL-C TG, ApoA1 and ApoB in ASGR1^+/- pigs and WT controls receiving a standard diet. Points indicate data from individual pigs (n = 8 for ASGR1^+/- pigs, n = 10 for WT controls. Data represent two independent experiments combined). P values are shown for indicated comparisons by the Student’s t-test. Values are mean ± SEM. The underlying data for this figure can be found in S1 Data. ApoA1, apolipoprotein A1; ApoB, apolipoprotein B; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; non-HDL-C, non-high-density lipoprotein cholesterol; TC, total cholesterol; TG, triglycerides; WT, wild-type.

Fig 3. ASGR1^+/- pigs are protected from HFHC diet-induced hypercholesterolemia and atherosclerosis.

Six-month-old ASGR1^+/- pigs and age-matched WT pigs were fed an HFHC diet for six months. A. Schematic diagram of pig treatment. B-F. Plasma levels of (B) non-HDL-C, (C) LDL-C, (D) TC, (E) TG and (F) VLDL-C at the indicated time points (n = 3 per group). G. Representative pictures of Sudan-IV-stained aortas showing atherosclerotic lesions in WT pigs but not in ASGR1^+/- pigs. H, I. (H) The gross lesion area and (I) the percentage of the Sudan-IV-stained area were measured by Image J software (n = 3 per group). J. Representative histological images of aortas from the aortic arch of WT and ASGR1^+/- pigs. Serial cuts from paraffin sections were stained with H&E and EVG, the black arrows point to the internal elastic lamina. P values are shown for indicated comparisons by the Student’s t-test. Values are mean ± SEM. The underlying data for this figure can be found in S1 Data. EVG, elastic van Gieson; H&E, hematoxylin-eosin; HFHC, high-fat and high-cholesterol; LDL-C, low-density lipoprotein cholesterol; non-HDL-C, non-high-density lipoprotein cholesterol; TC, total cholesterol; TG, triglycerides; VLDL-C, very-low-density lipoprotein cholesterol; WT, wild-type.

Fig 4. ASGR1 deficiency reduces hepatic cholesterol synthesis by downregulating HMGCR and augments cholesterol clearance by upregulating hepatic LDLR.

A. Hepatic transcriptome profiling of ASGR^-/- pigs and WT controls. The volcano diagram shows the differentially expressed genes (fold change > 2 and FDR < 0.05; downregulated in blue and upregulated in red) upon ASGR1 deficiency, in which cholesterol metabolism-related genes were labeled in purple. The blue dashed line indicates the threshold for significance (FDR <0.05), and the black dashed lines indicate the threshold for fold change (fold change > 2). B. Top ranking functional terms enriched for differentially expressed genes using the Metascape tool. GO-BP: biological process (blue) and DisGeNET (red). C. Relative mRNA expression of HMGCR and LDLR in livers of 24-month-old WT and ASGR1^-/- pigs, points indicate data from individual pigs (n = 5 per group, data represent two independent experiments combined). D, E. (D) Representative western blotting and (E) quantification of HMGCR and LDLR in livers of pigs (n = 3 per group, data are representative of two independent experiments). F. Rate of cholesterol biosynthesis in hepatocytes from 24-month-old WT and ASGR1^-/- pigs (n = 5 per group, data represent two independent experiments combined). G. Representative rate of cholesterol biosynthesis in HepG2 cells with ASGR1 knockdown by siRNA (n = 6 per group, data are representative of three independent experiments). H. Plasma lathosterol in ASGR1-deficient pigs and WT controls (n = 5 per group, data represent two independent experiments combined). I, J. (I) Liver total cholesterol (TC) and (J) total triglycerides (TG) content of pigs at 24 months (n = 5 per group, data represent two independent experiments combined). K, L. Changes in plasma (K) TC and (L) TG levels after Poloxamer-407 injection in pigs (n = 8 per group, data represent two independent experiments combined). M. Clearance of LDL-C in 12-month-old WT and ASGR1^+/–pigs (n = 6 per group, data represent two independent experiments combined). N, O. (N) Representative confocal immunofluorescence microscopy images of Dil-LDL uptake in HepG2 cells transfected with si-control and si-ASGR1 from three independent experiments. Confocal microscopic images represent the fluorescence intensity of Dil-LDL (red) and DAPI (blue). Scale bars, 100 μm. (O) Representative fluorescence quantification of isopropanol-extracted DiI-LDL (520 nm) (n = 3 per group, data are representative of three independent experiments). P values are shown. For analyses of intergroup differences between two groups, data were assessed using the Student’s t-test. For analysis of multiple comparisons, data were analyzed using ordinary one-way ANOVA followed by Tukey’s test. Values are mean ± SEM. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; DAPI, 4′, 6-diamidino-2-phenylindole; FDR, False Discovery Rate; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; LDL-C, low-density lipoprotein cholesterol; LDLR, low-density lipoprotein receptor; TC, total cholesterol; TG, triglycerides; WT, wild-type.

Fig 5. ASGR1-deficient pigs show high plasma levels of liver injury indicators.

A-F. Plasma levels of (A) ALP, (B) ALT, (C) AST, (D) GGT, (E) vitamin B12 and (F) ALB in ASGR1^-/- pigs and age-matched WT pigs, as well as in ASGR1^+/- pigs. Points indicate data from individual pigs, at the indicated time points (n = 6 for ASGR1^-/- pigs, n = 8 for ASGR1^+/- pigs, n = 10 for WT pigs, data represent two independent experiments combined). P values are shown for indicated comparisons by the Student’s t-test. Values are mean ± SEM. The underlying data for this figure can be found in S1 Data. ALB, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aminotransferase; GGT, gamma glutamyl transpeptidase; WT, wild-type.

Fig 6. ASGR1-deficient pigs show liver injury.

A, B. (A) Representative histological images of the TUNEL assay on liver tissue of 24-month-old WT and ASGR1-deficient pigs (Scale bars, 50 μm, 100 μm), (B) the apoptotic index quantification in livers of 24-month-old WT and ASGR1-deficient pigs (n = 5 per group, data represent two independent experiments combined). C. Relative mRNA expression of ER stress marker genes ATF4 and CHOP in livers of pigs (n = 5 per group, data represent two independent experiments combined). D, E. Representative (D) Western blot analysis and (E) quantification of ATF4 and CHOP in the livers of pigs (n = 3 per group, data are representative of two independent experiments). F. Representative the fluorescence microscopy images of HepG2 cells treated with vehicle, 100 nM ASGR1-siRNA or 100 nM ASGR1-siRNA plus 400 μM TUDCA for 24 h, were stained with AV-FITC (green) and PI (red), and observed by confocal microscopy (×400). G, H. Quantification of the (G) AV-FITC positive cells and (H) PI positive nuclei compared with total HepG2 cells. Values are means ± SEM, (n = 3 per group, data are representative of three independent experiments). I. H&E and Masson’s trichrome staining of liver sections from 24-month-old pigs fed a normal diet. H&E staining showing inflammation (arrow) and Masson’s trichrome staining of the distribution of collagen in blue demonstrated positive fibrosis in the livers of ASGR1-deficient pigs (Scale bars, 50 μm, and 100 μm). J, K. Relative mRNA expression of (J) inflammation and (K) fibrosis-related markers in livers of ASGR1-deficient pigs and WT controls (n = 5 per group, data represent two independent experiments combined). P values are shown for indicated comparisons were analyzed using ordinary one-way ANOVA followed by Tukey’s test. Values are mean ± SEM. Points indicate data from individual pigs. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; ATF4, Activating Transcription Factor 4; AV-FITC, Apoptosis determined by Annexin V fluorescein isothiocyanate; CHOP, C/EBP homologous protein; ER, Endoplasmic reticulum; H&E, hematoxylin-eosin; PI, propidium iodide; TUDCA, tauroursodeoxycholic acid; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; WT, wild-type.

Fig 7. Graphical summary of the underlying mechanisms of ASGR1 deficiency-mediated atheroprotective effects and liver injury.

Left, ASGR1 deficiency prevents atherosclerosis by downregulation of HMGCR-catalyzed hepatic cholesterol synthesis and its subsequent VLDL secretion, as well as by upregulation of LDLR-mediated hepatic LDL uptake. Right, the occurrence of liver injury shown by elevated hepatic enzymes, extensive apoptosis, mild inflammation and fibrosis due to ER stress-induced apoptosis upon ASGR1 deficiency.