LCAT protects against Lipoprotein‐X formation in a murine model of drug‐induced intrahepatic cholestasis

Figures

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  Figure 1. Changes in hepatic function following treatment with ANIT. Mice (WT, LCAT‐Tg, and LCAT‐KO, males, n = 5 per group) were fasted for 4‐6 hours and then bled by retro‐orbital sinus blood collection (~75‐100 μL). Mice were bled before, 1, 2, 3, and 4 days after ANIT gavage via the retro‐orbital sinus. Hepatic function markers, Total Bilirubin (A), Direct Bilirubin (B), Indirect Bilirubin (C), ALT (D), AST (E), and alkaline phosphatase (F.) were measured as described in the Methods section. *P<.05 compared to WT and LCAT‐Tg mice, # P<.05 compared to LCAT‐Tg mice

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  Figure 2. Change in plasma lipid and lipoproteins following ANIT treatment. Mice (WT, LCAT‐Tg, and LCAT‐KO, males, n = 4 per group) were fasted for 4‐6 hours and then bled by retro‐orbital sinus blood collection (~75‐100 μL). Mice were bled before, 1, 2, and 3 days after ANIT gavage via the retro‐orbital sinus, until peak levels of Lp‐X were present. Total Cholesterol (A), Free Cholesterol (B), Triglycerides (C), Phospholipids (D), Cholesteryl Ester (E) were measured as described in the Methods section. (F) surface/core lipid ratio, was calculated following the formula (FC + PL)/(CE + TG). (G) LCAT activity, was measured using endogenous substrate and calculated as described in the Methods section; red line represents the LCAT‐Tg activity (red legend, right, red x‐axis) while blue and black lines represent LCAT‐KO and control mice, respectively (black legend, left x‐axis). *P<.05 increase over baseline, # P<.05 decrease over baseline

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  Figure 3. Plasma lipoproteins by agarose gel electrophoresis. Plasma was obtained from mice (WT, n = 3, LCAT‐Tg, n = 3and LCAT‐KO, n = 3, males) either before (Baseline) or 1, 2, or 3 days after ANIT treatment (a single gavage of 100 mg/kg). 10 μL of plasma were loaded onto duplicate agarose gels followed by electrophoresis for 1 hour at 100 V. Gels were stained with Sudan Black (upper panels) or Filipin (lower panels) as described in Methods section. 10 μL of purified human LDL, VLDL, and HDL were included as standards loaded on the left side of each gel. Lp‐X can be seen as backwards‐running (ie cathode‐migrating) fluorescent material by Filipin staining. The origin (point of application) is marked by a red arrow at the left of the gels and by a thin blue line running across the center of the gels. Vertical red lines to the left of the gels mark the regions where HDL, VLDL, LDL, and Lp‐X migrate. HDL, LDL, and VLDL migrate towards the anode (+) and Lp‐X migrates toward the cathode (−)

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  Figure 4. Densitometric quantification of Lp‐X in Filipin stained gels. The fluorescent cathode‐migrating Lp‐X signal for samples on each gel were normalized to the fluorescent purified LDL signal on each gel to ensure accurate comparisons between gels. Average Lp‐X intensities (normalized to LDL) for each mouse line plus minus 1 SD are shown as a function of days after the ANIT treatment. WT; n = 3, LCAT‐KO; n = 3 and LCAT‐Tg; n = 3

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  Figure 5. Lp‐X in WT and LCAT‐KO mouse liver. Transmission electron microscopy reveals the appearance of multilamellar Lp‐X in WT and LCAT‐KO mouse liver compartments. Lp‐X is taken up by Kupffer cells in WT (A, n = 40 slices) and LCAT‐KO (C, n = 12 slices) mouse liver and is seen in hepatic sinusoids of LCAT‐KO mice (E). Regions denoted by white boxes in (A), (C), and (E) are shown at higher magnification in (B), (D), and (F), respectively. Scale bars: (A): 2 μm; (B) 600 nm; (C): 800 nm; (D): 400 nm; (E): 1 μm; (F): 500 nm

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  Figure 6. In vivo treatment with rhLCAT reduces Lp‐X in ANIT‐induced cholestasis. WT mouse plasma electrophoresis at baseline (A), on day 3 after ANIT gavage and on day 3 after ANIT gavage with multiple in vivo injections of rhLCAT (B). Plasma of six WT mice (n = 6) is shown in (A). Following electrophoresis gels were staining with Filipin (top panels) or Sudan Black (bottom panels) as described in Figure 3. (A) lanes 1, 2 reflect the migration of purified human LDL and VLDL, respectively; lanes 3‐8, WT mouse plasma of distinctive mice. (B) lanes 1‐3 reflect the migration of purified human LDL, VLDL, and HDL respectively, lanes 4‐6 reflect plasma from three individual WT mice plasma on day 3 after ANIT gavage; lanes 7‐9 show the remodeling effect produced by treatment with rhLCAT, in vivo, on mouse plasma, on day 3 after ANIT gavage. Lp‐X is visible by Filipin and, Sudan staining (mouse m3), in lanes 4‐6 below the red (origin, point of application) line. LDL, VLDL, and HDL migrate toward the anode (+, top) and Lp‐X migrates toward the cathode (−, bottom)

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  Figure 7. Recombinant human LCAT injection reduces Lp‐X in cholestatic murine plasma. FPLC analysis of WT mouse plasma at baseline (A and B), on day 3 after ANIT gavage (C and D) and on day 3 after ANIT gavage with multiple injections of rhLCAT in vivo (E and F). One hundred and fifty microliters of mouse plasma were separated through two Superose 6 columns in series producing 60 (0.5 mL each) fractions. Each fraction was analyzed for its cholesterol (total [TC] or unesterified [FC]) and phospholipid [PL] content as described in Methods section. LDL, VLDL, and HDL migrate toward the anode (+, top) and Lp‐X migrates toward the cathode (−, bottom; B, D, and F). Typically, VLDL elutes in fractions 8‐22, LDL elutes in fractions 23‐32 and HDL elutes in fractions 35‐60 (A, C, and E). (A, C, E) illustrates the TC, FC, and PL profile of a WT mouse (A), WT mouse plasma on day 3 after ANIT gavage (C) and WT mouse plasma on day 3 after ANIT gavage followed by multiple in vivo injections of rhLCAT (E). (B, D, F) illustrates the corresponding migration of fractions 8‐32 (VLDL and LDL) on an agarose gel stained with Filipin as described in Figure 3. Lp‐X is visible in fractions 15‐35 on C and below the…