Amelioration of atherosclerosis in apolipoprotein E-deficient mice by combined RNA interference of lipoprotein-associated phospholipase A2 and YKL-40

Abstract

To test the hypothesis that combined RNA interference (RNAi) of lipoprotein-associated phospholipase A2 (Lp-PLA2) and YKL-40 is superior to RNAi of Lp-PLA2 or YKL-40 alone in ameliorating atherosclerosis. A total of 120 apolipoprotein E-deficient mice (apoE-/- mice) were randomly divided into five groups, including the vehicle alone, scrambled RNAi, Lp-PLA2 RNAi, YKL-40 RNAi, and combined Lp-PLA2 and YKL-40 RNAi groups. Constrictive collars were used to induce plaque formation. Lp-PLA2 RNAi and YKL-40 RNAi viral suspensions were transduced into carotid plaques of the mice. Carotid plaques were harvested for histological analysis four weeks after viral vector transduction. Inflammatory gene expression in the plasma and atherosclerotic plaques was determined by ELISA and real-time PCR. Four weeks after RNAi, the serum concentration and plaque mRNA expression of Lp-PLA2 and YKL-40 were remarkably attenuated, leading to reduced inflammatory gene expression. Plaques from the Lp-PLA2 or YKL-40 RNAi group showed lower lipid content, higher collagen content, increased fibrous cap thickness, and lower mRNA expressions of MCP-1 and MMP-8 than than those in the vehicle and scramble groups. When compared with the isolated Lp-PLA2 or YKL-40 RNAi group, the combined Lp-PLA2 and YKL-40 RNAi group exhibited higher collagen content and fibrous cap thickness, and lower lipid content and local inflammation. The beneficial effects of RNAi were independent of the plasma lipoprotein profile. Combined RNAi of Lp-PLA2 and YKL-40 is superior to RNAi of Lp-PLA2 or YKL-40 alone in ameliorating atherosclerosis.

Fig 1. Silencing of YKL-40 in RAW264.7 cells by lentiviral vector-mediated YKL-40 shRNAs.

RAW264.7 cells were transduced with 50 MOI of 4 different kinds of shRNA vector, and YKL-40 expression was measured on day 4 by real-time PCR following transduction. GAPDH was used as an internal control. (A) YKL-40 mRNA expression was detected by real-time PCR (n = 8, *P<0.05). (B) RNAi inhibited the expressions of MCP-1 in RAW264.7 cells (n = 8). (C) RNAi inhibited the expression of MMP-8 in RAW264.7 cells (n = 8). No significant difference was found between the vehicle and scrambled groups. Vehicle = vehicle group; Scrambled = scrambled group (negative control). “-” and “+” indicate the absence and presence of lentivirus, respectively. Data are shown as the mean values ± SD obtained from triplicate experiments. *P<0.05 vs. vehicle groups; ^▲P>0.05 vs. vehicle group.

Fig 2. Knockdown of Lp-PLA₂ and YKL-40 in vivo.

(A) mRNA expression of Lp-PLA₂ in the plaques of the vehicle, scrambled, Lp-PLA₂ RNAi, YKL-40 RNAi, and combined Lp-PLA₂ and YKL-40 RNAi groups (n = 12 /group); (B) mRNA expression of YKL-40 in the plaques from all groups (n = 12 /group); (C) The plasma concentrations of Lp-PLA₂ in all groups (n = 24). (D) The plasma concentrations of YKL-40 in all groups (n = 24). Vehicle = vehicle group; scrambled = scrambled group (negative control). Lp-PLA₂ RNAi = Lp-PLA₂ RNAi group, YKL-40 RNAi = YKL-40 RNAi group, combined RNAi = combined Lp-PLA₂ and YKL-40 RNAi group. Data are shown as the mean values ± S.D. *P<0.05 vs. vehicle groups; ^▲P>0.05 vs. vehicle group.

Fig 3. Plaque morphology of the vehicle, scrambled, Lp-PLA₂ RNAi, YKL-40 RNAi, and combined Lp-PLA₂ and YKL-40 RNAi groups.

Cross-sections of plaques from all groups were stained with HE, ORO and Masson’s trichrome (n = 12). Magnification 200×.

Fig 4. Comparison of plaque morphology.

(A) Comparison of relative collagen content in the plaques of the vehicle, scrambled, Lp-PLA₂ RNAi, YKL-40 RNAi, and combined Lp-PLA₂ and YKL-40 RNAi groups (n = 12); (B) Comparison of relative lipid content in the plaques from all groups (n = 12). (C) Comparison of fibrous cap thickness in the plaques of all groups (n = 12). (D) Comparison of plaque area in the plaques of all groups (n = 12). *P<0.05 vs. vehicle groups; ^▲P>0.05 vs. vehicle group; ^△P<0.05 vs. Lp-PLA₂ RNAi and YKL-40 RNAi groups. ⁰P>0.05 vs. Lp-PLA₂ RNAi group.

Fig 5. Effects of RNAi on inflammation markers in vivo.

(A) mRNA expression of MCP-1 in the plaques of the vehicle, scrambled, Lp-PLA₂ RNAi, YKL-40 RNAi, and combined Lp-PLA₂ and YKL-40 RNAi groups (n = 12); (B) mRNA expression of MMP-8 in the plaques from all groups (n = 12); (C) The concentrations of MCP-1 in the plasma from all groups (n = 24). (D) The concentrations of MMP-8 in the plasma from all groups (n = 24). *P<0.05 vs. vehicle groups; ^▲P>0.05 vs. vehicle group; ^△P<0.05 vs. Lp-PLA₂ RNAi and YKL-40 RNAi groups.