Cholesterol-lowering drugs cause dissolution of cholesterol crystals and disperse Kupffer cell crown-like structures during resolution of NASH - PubMed (original) (raw)

Cholesterol-lowering drugs cause dissolution of cholesterol crystals and disperse Kupffer cell crown-like structures during resolution of NASH

George N Ioannou et al. J Lipid Res. 2015 Feb.

Abstract

Cholesterol crystals form within hepatocyte lipid droplets in human and experimental nonalcoholic steatohepatitis (NASH) and are the focus of crown-like structures (CLSs) of activated Kupffer cells (KCs). Obese, diabetic Alms1 mutant (foz/foz) mice were a fed high-fat (23%) diet containing 0.2% cholesterol for 16 weeks and then assigned to four intervention groups for 8 weeks: a) vehicle control, b) ezetimibe (5 mg/kg/day), c) atorvastatin (20 mg/kg/day), or d) ezetimibe and atorvastatin. Livers of vehicle-treated mice developed fibrosing NASH with abundant cholesterol crystallization within lipid droplets calculated to extend over 3.3% (SD, 2.2%) of liver surface area. Hepatocyte lipid droplets with prominent cholesterol crystallization were surrounded by TNFα-positive (activated) KCs forming CLSs (≥ 3 per high-power field). KCs that formed CLSs stained positive for NLRP3, implicating activation of the NLRP3 inflammasome in response to cholesterol crystals. In contrast, foz/foz mice treated with ezetimibe and atorvastatin showed near-complete resolution of cholesterol crystals [0.01% (SD, 0.02%) of surface area] and CLSs (0 per high-power field), with amelioration of fibrotic NASH. Ezetimibe or atorvastatin alone had intermediate effects on cholesterol crystallization, CLSs, and NASH. These findings are consistent with a causative link between exposure of hepatocytes and KCs to cholesterol crystals and with the development of NASH possibly mediated by NLRP3 activation.

Keywords: caspase 1; crown-like structure; lipotoxicity; nonalcoholic steatohepatitis.

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Figures

Fig. 1.

Treatment with ezetimibe and atorvastatin eliminates hepatic cholesterol crystals and ameliorates inflammation and fibrosis. Liver sections of foz/foz mice on an atherogenic diet treated with “vehicle” (control), ezetimibe, or atorvastatin or with a combination of both ezetimibe and atorvastatin (“combination”) and stained with hematoxylin and eosin (H&E), Sirius red (for collagen), anti-F4/80 antibodies (for macrophages), or unstained frozen sections viewed with polarized light for birefringent cholesterol crystals. H&E or Sirius red stained liver sections demonstrate that foz/foz mice on an atherogenic diet treated with “vehicle” (control) developed fibrosing NASH. In contrast, foz/foz mice treated with combination of ezetimibe and atorvastatin show resolution of fibrotic NASH. Anti-F4/80 staining, which identifies macrophages, shows multiple rings of macrophages forming CLSs as they surround hepatocyte lipid droplets in the vehicle-treated mice. In the ezetimibe- and/or atorvastatin-treated mice, mostly scattered isolated macrophages were seen, with rare CLSs in the ezetimibe- or atorvastatin-treated mice and none in the mice treated with both drugs. Frozen liver sections viewed with polarized light demonstrate birefringence within a large proportion of lipid droplets in the “vehicle”-treated group (control), including droplets that are entirely birefringent appearing as a “Maltese cross.” There was progressively less birefringence in the atorvastatin-treated and ezetimibe-treated groups and almost no birefringence in the group that was treated with both drugs. This birefringence is due to cholesterol crystallization as demonstrated by filipin staining in Figure 3. Black scale bars are 50 µm; white scale bars are 100 µm. IHC, immunohistochemistry.

Fig. 2.

Birefringent material within hepatocyte lipid droplets stains with filipin, demonstrating the presence of cholesterol crystals. Frozen liver sections from foz/foz mice fed a high-fat, high-cholesterol diet for 24 weeks, with the last 8 weeks supplemented with vehicle (A, B), ezetimibe (C, D), atorvastatin (E, F), or both ezetimibe and atorvastatin (G, H). The sections are stained with filipin (blue) to identify free cholesterol. Each section is viewed with polarized light (to demonstrated cholesterol crystals) or fluorescent light (to demonstrate filipin). Mice supplemented with vehicle, which developed NASH, had strongly birefringent crystals within a large proportion of lipid droplets (A) that also stain with filipin (B), demonstrating that they represent cholesterol crystals. Mice supplemented with both ezetimibe and atorvastatin, in which NASH was almost completely ameliorated, did not have any hepatic cholesterol crystals (G, H). Mice supplemented with only atorvastatin or only ezetimibe, in which NASH was only partly ameliorated, had some evidence of hepatic cholesterol crystals, especially in the atorvastatin group, but reduced as compared with the group that received both drugs.

Fig. 3.

KCs aggregate around hepatocytes that contain cholesterol crystals forming crown-like structures, which disperse after treatment with ezetimibe and atorvastatin. Frozen liver sections are shown from foz/foz mice fed a high-fat, high-cholesterol diet for 24 weeks, with the last 8 weeks supplemented with vehicle (A, B), ezetimibe (C, D), atorvastatin (E, F), or both ezetimibe and atorvastatin (G, H). The sections are stained with fluorescently labeled anti-TNFα antibodies (green) to identify activated KCs. Each section is viewed with either polarized light (to demonstrate cholesterol crystals) or fluorescent light (to demonstrate anti-TNFα). Mice supplemented with vehicle, which developed NASH, had strongly birefringent crystals within a large proportion of lipid droplets (A) that were surrounded by crown-like structures of activated macrophages (B). Mice supplemented with both ezetimibe and atorvastatin, in which NASH was almost completely ameliorated, did not have any hepatic cholesterol crystals (G) or macrophage crown-like structures (H). Mice supplemented with only atorvastatin or only ezetimibe, in which NASH was only partly ameliorated, had some evidence of hepatic cholesterol crystals and CLSs, especially in the atorvastatin group, but this amount was reduced as compared with the group that received both drugs.

Fig. 4.

KCs in CLS surrounding cholesterol crystal-containing remnant lipid droplets demonstrate activation of the NLRP3 inflammasome. Frozen liver sections are shown from foz/foz mice fed a high-fat, high-cholesterol diet for 24 weeks. Each section is viewed with polarized light (to demonstrate cholesterol crystals) or fluorescent light. A and B: KCs from vehicle-treated mice stained with anti-NLRP3 (identifies the NLRP3 inflammasome, colored green) showing that the macrophages that aggregate around heavily crystallized droplets forming CLSs exhibit critical components of the NLRP3 inflammasome. C and D: KCs from mice treated with both ezetimibe and atorvastatin stained with fluorescently labeled anti-NLRP3 and showing no staining. E and F: KCs from vehicle-treated mice stained with FLICA caspase 1, which identifies activated (cleaved) caspase 1 (red), showing that the macrophages in CLSs expressed activated caspase 1 and can potentially cleave pro-IL1 into IL1. G and H: KCs from mice treated with both ezetimibe and atorvastatin showing no staining. I, J, and K: mRNA expression levels (mean ± SEM) for components of the NLRP3 inflammasome (Caspase 1, Nalp3, and Asc, respectively) demonstrating reduced expression in mice treated with ezetimibe and atorvastatin relative to vehicle-treated mice (this did not reach statistical significance defined as P < 0.05).

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Cholesterol-lowering drugs cause dissolution of cholesterol crystals and disperse Kupffer cell crown-like structures during resolution of NASH - PubMed (original) (raw)