Differential Expression of the Host Lipid Regulators ANGPTL-3 and ANGPTL-4 in HCV Infection and Treatment

Figures

• 

  Figure A1. Proliferation assay in early HCV infection. The CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega) was used to assess proliferation rates of HCV-infected vs. mock-infected Huh7.5 hepatoma cells at the given time points p.i. The test was carried out according to the manufacturer’s instructions, and the absorbance recorded at 490 nm represents cell growth in the absence (dotted line) or presence (continuous line) of HCV. The experiment was carried out twice in triplicate and did not demonstrate significant differences between the HCV- and the mock-infected cell populations with time.

• 

  Figure 1. The role of angiopoietin-like proteins (ANGPTLs) in lipid metabolism. ANGPTL-3 and ANGPTL-4 regulate serum lipid levels via inhibition of the enzymatic activities of lipoprotein lipase (LPL), hepatic lipase (HL) and endothelial lipase (LIPG) (LIPG is regulated by ANGPTL-3 only). Lipases hydrolyse the triacylglycerols (TAG) of lipoproteins and chylomicrons into non-esterified free fatty acids (FFA) and monoacylglycerols. Upon silencing of ANGPTLs, loss of lipase regulation results in enhanced lipolysis and increased clearance of serum lipids by the liver and other tissues [43], eventually leading to hypolipidaemia. On the contrary, overexpression of ANGPTLs blocks lipases, thereby increasing circulating lipoproteins and resulting in hyperlipidaemia [25,26].

• 

  Figure 2. ANGPTL-3 and ANGPTL-4 serum levels throughout the natural history of HCV infection. Clinical samples of HCV patients from all stages of liver disease were used for the determination of ANGPTL-3 (a) and ANGPTL-4 (b) serum concentrations by ELISA. The black horizontal lines depict the median value of the respective group. Statistically significant differences within groups are denoted by one star (p-value ≤ 0.05), or two stars (p-value ≤ 0.005).

• 

  Figure 3. mRNA expression levels of ANGPTL-3 and ANGPTL-4 during long-term HCV infection in vitro. Huh7.5 hepatoma cells infected with HCV-3a were harvested at the stated time points post-infection (p.i.) and subjected to RT-qPCR with (a) ANGPTL-3- or (b) ANGPTL-4-specific oligonucleotide primers. HCV NS3-3a mRNA expression was used as an indicator of HCV replication (black continuous line on a logarithmic axis). Each bin represents the ratio of the relative mRNA expression in HCV-infected cells to that of the mock-infected control cells (dotted line). Statistically significant differences within groups are denoted by one star (p-value ≤ 0.05), or two stars (p-value ≤ 0.005).

• 

  Figure 4. (a) HCV-3a replication levels before and after HCV eradication in an in vitro HCV clearance model. Huh7.5 hepatoma cells were infected with HCV-3a and treated with a mixture of HCV antiviral compounds (DAA) or left untreated. The infected cells were harvested at the stated time points p.i. and subjected to RT-qPCR with NS3-3a-specific oligonucleotide primers as an indicator of HCV replication in the presence (continuous line) and absence (dotted line) of the antiviral treatment. Relative mRNA expression for the HCV NS3-3a gene is plotted on a logarithmic axis. Statistically significant differences between the HCV NS3-3a mRNA values from DAA-treated and untreated infected cells are denoted by one star (p-value ≤ 0.05), or two stars (p-value ≤ 0.005). (b) HCV infectivity following DAA administration in an in vitro HCV clearance model. Supernatants from the HCV-3a-infected hepatoma cell cultures described above were collected before (white bars) and after (grey bars) DAA treatment and used for the isolation of viral RNA. Statistically significant differences within groups are denoted by one star (p-value ≤ 0.05), or two stars (p-value ≤ 0.005).

• 

  Figure 5. mRNA expression levels of ANGPTL-3 and ANGPTL-4 before and after HCV eradication in an in vitro HCV clearance model. Huh7.5 hepatoma cells infected with HCV-3a were harvested at the stated time points p.i. and subjected to RT-qPCR with (a) ANGPTL-3- or (b) ANGPTL-4-specific oligonucleotide primers. White and grey bins represent the ratio of relative mRNA expression in HCV-infected (p.i.) or HCV-cured (p.i. + DAA) cells to that of the mock-infected (m.i.) control (black bins). Statistically significant differences within groups are denoted by one star (p-value ≤ 0.05), or two stars (p-value ≤ 0.005).

• 

  Figure 6. Serum levels of the key profibrotic cytokine TGF-β in correlation with ANGPTL-3 and ANGPTL-4 in HCV patients with mild fibrosis, advanced fibrosis and cirrhosis. The naturally active form of TGF-β (white box plots) was determined by sandwich ELISA from the sera of HCV patients prior to DAA treatment and is plotted together with ANGPTL-3 (light grey box plots) and ANGPTL-4 (dark grey box plots) levels against fibrosis stage. Statistically significant differences within groups are denoted by one star (p-value ≤ 0.05), or two stars (p-value ≤ 0.005).