AAV-mediated hepatic LPL expression ameliorates severe hypertriglyceridemia and acute pancreatitis in Gpihbp1 deficient mice and rats

Abstract

GPIHBP1 plays an important role in the hydrolysis of triglyceride (TG) lipoproteins by lipoprotein lipases (LPLs). However, Gpihbp1 knockout mice did not develop hypertriglyceridemia (HTG) during the suckling period but developed severe HTG after weaning on a chow diet. It has been postulated that LPL expression in the liver of suckling mice may be involved. To determine whether hepatic LPL expression could correct severe HTG in Gpihbp1 deficiency, liver-targeted LPL expression was achieved via intravenous administration of the adeno-associated virus (AAV)-human LPL gene, and the effects of AAV-LPL on HTG and HTG-related acute pancreatitis (HTG-AP) were observed. Suckling Gpihbp1^-/- mice with high hepatic LPL expression did not develop HTG, whereas Gpihbp1^-/- rat pups without hepatic LPL expression developed severe HTG. AAV-mediated liver-targeted LPL expression dose-dependently decreased plasma TG levels in Gpihbp1^-/- mice and rats, increased post-heparin plasma LPL mass and activity, decreased mortality in Gpihbp1^-/- rat pups, and reduced the susceptibility and severity of both Gpihbp1^-/- animals to HTG-AP. However, the muscle expression of AAV-LPL had no significant effect on HTG. Targeted expression of LPL in the liver showed no obvious adverse reactions. Thus, liver-targeted LPL expression may be a new therapeutic approach for HTG-AP caused by GPIHBP1 deficiency.

Keywords: GPIHBP1; acute pancreatitis; adeno-associated virus; gene therapy; hypertriglyceridemia; lipoprotein lipase.

Graphical abstract

Figure 1

Correlation between Lpl mRNA expression and plasma lipid levels in suckling liver of Gpihbp1^−/− rats and mice (A and B) Lpl mRNA expression in the liver of Gpihbp1^−/− mice (n = 6) and rats (n = 6) during suckling period. (C) Log2 of plasma TG and TC levels in WT (n = 4) and Gpihbp1^−/− mice (n = 6) during suckling period. (D) Log2 of plasma TG and TC levels in WT (n = 4) and Gpihbp1^−/− rats (n = 6) during suckling period. Data are presented as mean ± standard error of mean (SEM). Statistical comparisons were made using two-way ANOVA with Sidak correction. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. WT mice or WT rats.

Figure 2

Construction and infection efficacy to liver of AAV-LPL recombinant viral vector (A) Structure diagram of AAV-LPL recombinant viral vector. (B) Representative fluorescent images of livers from WT mice treated with different doses of AAV-LPL (n = 6 per group). Scale bar, 100 μm. (C) Representative fluorescent images of various organs from WT mice treated with 1 × 10¹³ vg/kg AAV-EGFP (n = 6). Scale bar, 100 μm. (D) Expression of human LPL mRNA relative to Gapdh in various tissues of WT mice treated with 1 × 10¹³ vg/kg AAV-LPL (n = 6). (E) Expression of human LPL mRNA relative to Gapdh in liver from WT mice treated with different doses of AAV-LPL (n = 6 per group). (F) Immunohistochemical analysis of LPL in the liver from WT mice treated with different doses of AAV-LPL (n = 6 per group). Scale bars, 200 and 50 μm. Data are presented as mean ± SEM.

Figure 3

Comparison of lipid-lowering effects of AAV-LPL-S447X and AAV-LPL-WT in Gpihbp1^−/− mice (A and B) Plasma TG and TC levels at 7 days (7d), 14 days (14d), and 28 days (28d) after treatment with AAV-LPL-S447X and AAV-LPL-WT (n = 6 per group). Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared to PBS group, two-way ANOVA with Sidak correction.

Figure 4

Effect of intravenous injection of AAV-LPL on plasma lipid levels in Gpihbp1^−/− mice (A and B) Dynamic changes of plasma TG and TC levels at different time points after different doses of AAV-LPL treatment. (C) Plasma TG and TC levels at 2 months after different doses of AAV-LPL treatment. (D and E) Post-heparin plasma LPL concentration and activity after 2 months of treatment with different doses of AAV-LPL. (F) Representative plasma sample appearance after 2 months of treatment with different doses of AAV-LPL. PBS, n = 6; 1 × 10¹³ vg/kg AAV-EGFP, n = 6; 1 × 10¹¹ vg/kg AAV-LPL, n = 12; 1 × 10¹³ vg/kg AAV-LPL, n = 12. For (A–C), statistical comparisons were made using two-way ANOVA with Sidak correction. For (D) and (E), statistical comparisons were made using one-way ANOVA with LSD correction. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. PBS or 1 × 10¹³ vg/kg AAV-EGFP group.

Figure 5

Effect of intravenous injection of AAV-LPL on survival rate and plasma lipid levels in Gpihbp1^−/− rats (A) Experimental protocol of the administration of AAV-LPL in suckling and adult Gpihbp1^−/− rats. Neonatal Gpihbp1^−/− suckling pups were injected with 1 × 10¹³ vg/kg AAV5-LPL via orbital vein at 7 days of birth. Another serotype of AAV recombinant viral vector, AAV8-LPL, was administered in these animals at age 8 weeks in high (1 × 10¹³ vg/kg) and low dose (1 × 10¹¹ vg/kg), respectively. Gpihbp1^−/− rats receiving PBS served as controls. (B) Twenty-eight-day survival curves of suckling Gpihbp1^−/− rats treated with 1 × 10¹³ vg/kg AAV5-LPL (n = 52) or PBS (n = 44). (C and D) Dynamic changes of plasma TG and TC levels at different time points after AAV5-LPL and AAV8-LPL gene therapy. (E and F) Post-heparin plasma LPL concentrations and activities after 2 weeks (10 weeks old) of the administration of AAV8-LPL at different doses. (G) Representative plasma sample appearance after 2 weeks (10 weeks old) of the administration of AAV8-LPL at different doses. PBS, n = 12; 1 × 10¹³ vg/kg AAV5-LPL, n = 32; 1 × 10¹¹ vg/kg AAV8-LPL, n = 16; 1 × 10¹³ vg/kg AAV8-LPL, n = 16. Data are presented as mean ± SEM. Survival analysis was made using Kaplan-Meier method and log rank test. For (C) and (D), statistical comparisons were made using two-way ANOVA with Sidak correction. For (E) and (F), statistical comparisons were made using one-way ANOVA with LSD correction. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. PBS group.

Figure 6

Effect of intravenous administration of AAV-LPL on the pathogenesis of HTG-AP in Gpihbp1^−/− mice (A) Gpihbp1^−/− mice were injected intraperitoneally with a very low dose (5 μg/kg) of caerulein (Cae) after intravenous injection of 1 × 10¹³ vg/kg AAV-LPL, and the susceptibility to HTG-AP was observed by H&E staining of pancreatic tissue. WT + PBS, n = 4; Gpihbp1^−/− + PBS, n = 4; Gpihbp1^−/− + 1 × 10¹³ vg/kg AAV-LPL + PBS, n = 4; WT + Cae, n = 6; Gpihbp1^−/− + Cae, n = 6; Gpihbp1^−/− + 1 × 10¹³ vg/kg AAV-LPL + Cae, n = 6. (B) Plasma amylase and lipase levels in Gpihbp1^−/− mice injected with very-low-dose (5 μg/kg) Cae-induced HTG-AP. (C) HTG-AP was induced by intraperitoneal injection of Cae (50 μg/kg) after intravenous injection of 1 × 10¹³ vg/kg AAV-LPL to Gpihbp1^−/− mice. Pancreatic injury was observed by H&E staining. WT + PBS, n = 4; Gpihbp1^−/− + PBS, n = 4; Gpihbp1^−/− + 1 × 10¹³ vg/kg AAV-LPL + PBS, n = 4; WT + Cae, n = 6; Gpihbp1^−/− + Cae, n = 6; Gpihbp1^−/− + 1 × 10¹³ vg/kg AAV-LPL + Cae, n = 6. (D) Changes in plasma amylase and lipase levels after induction of HTG-AP by 50 μg/kg of Cae. Scale bar, 50 μm. Data are presented as mean ± SEM. For (B) and (D), statistical comparisons were made using two-way ANOVA with Sidak correction. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. WT + Cae or Gpihbp1^−/− + Cae group.

Figure 7

Effect of intravenous administration of AAV-LPL on HFD-induced spontaneous HTG-AP in Gpihbp1^−/− rats (A) Experimental protocol of the administration of AAV-LPL on HFD-induced HTG-AP in Gpihbp1^−/− rats. Seven-day-old Gpihbp1^−/− suckling pups were injected with 1 × 10¹³ vg/kg AAV5-LPL, and subsequently AAV8-LPL was administered in these animals at age of 8 weeks in high (1 × 10¹³ vg/kg) and low dose (1 × 10¹¹ vg/kg), respectively. Two weeks after AAV8 treatment, Gpihbp1^−/− rats were fed with HFD to induce spontaneous HTG-AP. (B and C) The plasma TG and TC levels of Gpihbp1^−/− rats in each group at different time points after HFD feeding. (D) Representative H&E images of pancreatic and lung tissues of Gpihbp1^−/− rats in each group after 14 days of HFD feeding. WT + HFD, n = 8; Gpihbp1^−/− + PBS + HFD, n = 18; Gpihbp1^−/− + 1 × 10¹¹ vg/kg AAV8-LPL+HFD, n = 16; Gpihbp1^−/− + 1 × 10¹³ vg/kg AAV8-LPL + HFD, n = 16; Gpihbp1^−/− + chow diet (CD), n = 16. Scale bars, 100 and 25 μm. (E) Pathological score of pancreatic injury in Gpihbp1^−/− rats of each group after 14 days of HFD feeding. For (B) and (C), statistical comparisons were made using two-way ANOVA with Sidak correction. For (E), statistical comparisons were made using one-way ANOVA with LSD correction. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. Gpihbp1^−/− + PBS + HFD or Gpihbp1^−/− + 1 × 10¹¹ vg/kg AAV8-LPL + HFD.

Figure 8

Effect of intramuscular injection of AAV9-LPL recombinant viral vector on plasma lipid levels of Gpihbp1^−/− mice and rats (A) Dynamic changes of plasma TG and TC levels at different time points after intramuscular injection of AAV9-LPL in Gpihbp1^−/− mice (n = 6 per group). (B) Post-heparin plasma LPL concentration and activity after 2 months of AAV9-LPL treatment in Gpihbp1^−/− mice (n = 6 per group). (C) Muscular human LPL mRNA expression level after 2 months of AAV9-LPL treatment in Gpihbp1^−/− mice (n = 6 per group). (D) Dynamic changes of plasma TG and TC levels at different time points after intramuscular injection of AAV9-LPL in Gpihbp1^−/− rats (n = 6 per group). (E) Post-heparin plasma LPL concentrations and activities after 2 months of AAV9-LPL treatment in Gpihbp1^−/− rats (n = 6 per group). (F) Muscular human LPL mRNA expression level after 2 months of AAV9-LPL treatment in Gpihbp1^−/− rats (n = 6 per group). (G) Representative plasma sample appearance after 2 months of AAV9-LPL treatment in Gpihbp1^−/− mice and rats (n = 6 per group). (H) Immunofluorescence assay showed that liver LPL was colocalized with HSPG after 2 months of intravenous administration of AAV-LPL in Gpihbp1^−/− mice and rats (n = 6 per group). Scale bar, 100 μm. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. Gpihbp1^−/− + PBS or Gpihbp1^−/− + AAV9-EGFP group, one-way ANOVA and LSD test.