Soluble Endoglin as a Potential Biomarker of Nonalcoholic Steatohepatitis (NASH) Development, Participating in Aggravation of NASH-Related Changes in Mouse Liver

Figures

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  Figure 1. FFC diet and hsEng effects on body and liver weight as well as Alkaline phosphatase (ALP) and Alanine transaminase (ALT) activity. Increases in the bodyweight of each mouse were monitored at 1-week intervals throughout the 24 weeks of chow or FFC diet feeding (A). Bodyweight gain (B) and ratio liver/body weight (C) of mice after the feeding period. The activity of ALP (D) and ALT (E) in plasma. The data are presented as median with box and whiskers representing the interquartile range and 5th–95th percentiles (n = 8). * p < 0.05 ** p < 0.01, *** p < 0.001, using the Kruskal–Wallis test.

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  Figure 2. Human and mouse sEng levels in the blood, Eng, Klf6, and MMP14 expression in liver. Plasma levels of human sEng (A). Plasma levels of mouse sEng (B). Protein expression of MMP14 (C). mRNA expression of transcription factor Klf6 (D). Protein expression of mouse Eng (E). The data are presented as median with box and whiskers representing the interquartile range and 5th–95th percentiles (n = 8). * p < 0.05, ** p < 0.01, *** p < 0.001, using the Kruskal–Wallis test; † p < 0.05, using the Mann–Whitney test wild type mice fed with high-saturated fat, high-fructose high-cholesterol diet versus high human soluble endoglin mice fed with high-saturated fat, high-fructose, high-cholesterol diet (WTFFC–hsEngFFC).

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  Figure 3. Representative pictures of immunohistochemical staining for Eng, PECAM-1, and anti-α-smooth muscle actin (α-SMA) in mice liver. Eng expression (green) is marked by arrows (white) in WTchow (A), WTFFC (D) and hsEngFFC (G) mice. PECAM-1 expression (red) is marked by arrows (white) in WTchow (B), WTFFC (E) and hsEngFFC (H) mice. Nuclei staining in blue. α-smooth muscle actin (brown) is marked by arrows (black) in WTchow (C), WTFFC (F) and hsEngFFC (I) mice. Scale bar 100 µm.

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  Figure 4. Cholesterol levels in blood and cholesterol and TAG concentration in mice liver. Cholesterol concentration in plasma (A). The concentration of cholesterol (B) and TAG (C) in the liver. The data are presented as median with box and whiskers representing the interquartile range and 5th–95th percentiles (n = 8). * p < 0.05, ** p < 0.01, *** p < 0.001, using the Kruskal–Wallis test; † p < 0.05, †† p < 0.01, using the Mann–Whitney test (WTFFC–hsEngFFC).

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  Figure 5. Evaluation of steatosis, fibrosis, inflammation, and oxidative stress in the liver. Sirius red-stained liver sections [macrovesicular steatosis–black arrows and mild fibrosis–red arrows] (A). mRNA expression of genes involved in FA uptake (Cd36) (B), lipogenesis (Acaca, Fasn, Scd1) (C) and oxidation (Cpt1a, Pparα, Acacb, Ppargc1α) (D). mRNA expression of genes involved in lipid secretion (Mttp, Apob) (E). Gene expression of inflammatory (Il6), fibrogenic (Tgf-β1 , Acta2, Col1a1, Pdgfb) and oxidative stress (Nqo1, Hmox1) markers (F). The data are presented as median with box and whiskers representing the interquartile range and 5th–95th percentiles (n = 8). * p < 0.05, ** p < 0.01, *** p < 0.001, using the Kruskal–Wallis test; † p < 0.05, †† p < 0.01 using the Mann–Whitney test (WTFFC–hsEngFFC); § p = 0.065 using the Mann–Whitney test (WTchow–WTFFC).

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  Figure 6. Cholesterol metabolism in the liver in WT and hsEng mice. mRNA (A) and protein (B) expression of intake transporter for HDL cholesterol (Scrab1/Sr-b1), LDL influx transporter (Ldlr), efflux transporter of cholesterol to plasma (Abca1), the rate-limiting enzyme for cholesterol de novo synthesis (Hmgcr), and transporters for the elimination of cholesterol into bile (Abcg5/Abcg8) in the liver. The data are presented as median with box and whiskers representing the interquartile range and 5th–95th percentiles (n = 8). * p < 0.05, ** p < 0.01, *** p < 0.001, using the Kruskal–Wallis test.

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  Figure 7. Bile acid metabolism in the liver in WT and hsEng mice. Plasma concentration of BA (A). Bile flow rate (B). Biliary elimination of BA (C). mRNA (D) and protein (E) expression of transporters involved in BA uptake to the liver (Slc10a1/Ntcp), bile production (Abcb11/Bsep, Abcc2/Mrp2), and BA efflux to plasma (Abcc4/Mrp4). mRNA (F) and protein (G) expression of liver enzymes involved in BA synthesis from cholesterol (Cyp7a1, Cyp8b1, Cyp27a1). The data are presented as median with box and whiskers representing the interquartile range and 5th–95th percentiles (n = 8). * p < 0.05, ** p < 0.01, *** p < 0.001, using the Kruskal–Wallis test; † p < 0.05 using the Mann–Whitney test (WTFFC–hsEngFFC); § p = 0.053 using the Mann–Whitney test (WTchow–WTFFC).

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  Figure 8. Schematic representation of hsEng effects in lipid, cholesterol, and BA metabolic pathways in the NASH diet-induced mouse model. Increased hepatic TAG content may result from decreased beta-oxidation of fatty acids (via Accb and Cpt1) and its reduced efflux to plasma (by Apob and Mttp). Reduced conversion of cholesterol into BA (via Cyp7a1) may be the reason for increased cholesterol accumulation and consequent lower biliary elimination of BA in a NASH liver. Full red arrow—statistically significant. Dotted red arrow—borderline significance. Blue arrows—circulation of cholesterol. Green arrows—circulation of bile acids. Yellow arrow—efflux of glutathione. Orange arrows—circulation of TAG. Black arrows metabolic pathways. Abca1, ATP-binding cassette transporter; Abcg5/8, Dimer of ATP-binding cassette subfamily G member 5 and member 8; ACC, acetyl-CoA carboxylase; Apob, Apolipoprotein B; β-oxid, beta-oxidation; BA, bile acids; Bsep, Bile salt export pump; Cd36, cluster of differentiation 36; DAG, Diacylglycerol; DGAT, Diglyceride acyltransferase; FAS, Fatty acid synthase; FFA, free fatty acids; G-3-P, Glyceraldehyde-3-phosphate; GPAT, Glycerol-3-phosphate acyltransferase; GSH,…