Dual _targeting of hepatic fibrosis and atherogenesis by icosabutate, an engineered eicosapentaenoic acid derivative

2.1. Animals, treatments and analyses

2.2. Statistical analyses

Analyses were carried out by the respective institutions performing the studies. For the AMLN ob/ob model a two‐way ANOVA with Tukey's multiple comparisons test was performed for body weight and quantitative histological analyses. A one‐way ANOVA with Dunnett's post‐hoc test was used for all other parameters except hepatic gene expression for which a two‐tailed Student's t test was used (GraphPad Prism v7.03 software). For the APOE*3L.CETP studies statistical differences between groups were determined by using non‐parametric Kruskal‐Wallis followed by Mann‐Whitney U test for independent samples (SPSS software). For LX‐2 cell studies one‐way ANOVA with Dunnett's post‐hoc test was performed (GraphPad Prism v7.03 software). For hepatic lipidomics, differences between groups were tested using Student's t test (MassLynx 4.1 software). For all studies a P < .05 (≤.05 for APOE*3L.CETP studies) was considered statistically significant and all results are shown as mean ± standard error of mean (SEM).

3.1. Both icosabutate and OCA reduce hepatic steatosis, but only icosabutate reduces plasma ALT and hepatic macrophage numbers in AMLN ob/ob mice

In the AMLN ob/ob model neither icosabutate nor OCA affected bodyweight (Figure 1A). OCA significantly reduced liver weight by 19% (P < .05) at 8 weeks whereas icosabutate had no significant effect (Figure 1B). A dose‐dependent decrease in steatosis (Figure 1C) in response to icosabutate treatment was seen, with the 135 mpk dose achieving a 47% reduction while OCA reduced liver fat by 38% (both P < .001). Icosabutate also elicited a dose‐dependent effect on hepatic TAG lowering (Figure 1D), with reductions of 17, 35 and 40% (all P < .001), while OCA achieved a reduction of 16% (P < .05).

With respect to liver injury and inflammation, only icosabutate (90 and 135 mpk) reduced plasma ALT (−32 and −44% respectively, P < .001) (Figure 1E). To further assess the effects of either treatment on hepatic inflammatory macrophage infiltration, quantitative immunohistochemistry was performed to assess hepatic galectin‐3 (Gal‐3) content. All doses of icosabutate achieved a significant reduction in hepatic Gal‐3 (Figure 1F). Representative histological photomicrographs of liver cross sections stained with H&E and galectin‐3 are shown in Figure 1G. The decreases in Gal‐3 in response to icosabutate treatment occurred in conjunction with significant decreases in mRNA transcripts for key genes (eg TNF‐α, TGF‐β1, TGFRβ and CCR2) regulating hepatic inflammatory responses in AMLN ob/ob mice after 4 weeks treatment with icosabutate (FigureS1, section D).

3.2. Icosabutate prevents progression of hepatic fibrosis in AMLN ob/ob mice

To assess the effects of treatment with either icosabutate or OCA on fibrosis, hepatic concentrations of hydroxyproline (HYP) were measured via a biochemical assay in addition to type 1 collagen α1 (col1A1) protein content measured via quantitative immunohistochemistry. Icosabutate reduced hepatic col1A1 content expressed as % area at the 90 mpk dose by 27% (P < .01, Figure 2A) and as total col1A1 at both the 90 and 135 mpk doses (−32%, P < .01 and −23%, P < .05 respectively, Figure 2B). The study design employed a prebiopsy confirmation of fibrosis as an inclusion criterion. Change in col1A1 content (post‐biopsy minus prebiopsy, Figure 2C) demonstrates that all doses of icosabutate minimised the progression of fibrosis during the treatment period, albeit only the 90 mpk dose was significant (P < .01) vs vehicle. Representative histological photomicrographs of liver cross sections stained for col1A1 pre‐ or post‐treatment are shown in Figure 2D.

Only icosabutate (at the 90 and 135 mpk doses) significantly reduced HYP content expressed in both relative and total units with both the lowest icosabutate dose and OCA having no significant effect (Figure 2E and F respectively). In summary, the combined col1A1 and HYP data demonstrate that icosabutate effectively inhibits the progression of fibrosis.

The decreases in hepatic fibrosis in response to icosabutate treatment occurred in conjunction with significant decreases in mRNA transcripts for multiple genes regulating stellate cell activation, fibrogenesis and fibrolysis in AMLN ob/ob mice after 4 weeks treatment with icosabutate (Figure S1, section A).

3.3. Icosabutate reduces hepatic myofibroblast content in AMLN ob/ob mice in vivo and proliferative responses of human stellate (LX‐2) cells in vitro

To gain further insight into underlying drivers of the decrease in fibrosis with icosabutate treatment, α‐SMA content was measured as a marker of myofibroblast content. Icosabutate 90 and 135 mpk reduced α‐SMA content expressed both as % area and total (all P < .01, Figure 3A and B respectively) whereas OCA and icosabutate 45 mpk had no significant effect. Icosabutate 135 mpk also led to a significant reduction in post‐ vs prebiopsy α‐SMA content (Figure 3C). Representative histological photomicrographs of liver cross sections stained with α‐SMA pre‐ or post‐treatment are shown in Figure 3D. Overall, α‐SMA data demonstrate that icosabutate prevents the development of fibrosis in association with a decline in myofibroblast numbers.

In association with the finding of decreased myofibroblast content in response to treatment with icosabutate, we performed additional in vitro experiments in spontaneously proliferating LX‐2 cells, a widely used human hepatic stellate cell line. As fatty acids can serve as a fuel for proliferating stellate cells via autophagy, ²⁹ oleic acid (75 μmol/L) was included as a control. While no change in cell viability was observed at any dose, a significant reduction in LX‐2 cell proliferation was observed at the 25, 50 and 75 μmol/L icosabutate doses with the strongest inhibition (−33%, P < .001) seen at the 50 μmol/L dose (Figure 3E). These data suggest that the decrease in myofibroblast content in vivo may be driven by direct anti‐proliferative effects of icosabutate on myofibroblasts.

3.4. Icosabutate reduces hepatic lipotoxicity and downregulates the hepatic arachidonic cascade in AMLN ob/ob mice

In addition to lowering hepatic TAG, icosabutate (at 90 and 135 mpk) significantly decreased hepatic free fatty acids (FFAs, Figure 4A), diacylglycerols (DAG, Figure 4B) and ceramides (Figure 4C) whereas OCA significantly increased ceramides and reduced bile acids (Figure 4D) only.

Icosabutate (90 and 135 mpk) reduced HETEs (total 5‐, 11[R]‐ and 15[S]‐HETEs) by 45 and 51% respectively (P < .001) whereas both the lowest dose of icosabutate and OCA were without significant effect (Figure 4E). To assess if the marked decrease in HETEs could be mediated via decreased AA stores, concentrations of AA in the dominant membrane phospholipid, phosphatidylcholine (PC), were measured. As shown in Figure 4F icosabutate induced a marked and significant decrease in PC‐AA at the 90 mpk (−32%, P < .001) and 135 mpk (−42%, P < .001) doses while a small significant increase was seen in response to OCA treatment.

Expression of genes regulating hepatic HETE formation demonstrated a significant decrease in ALOX5AP (regulating 5‐HETE and LTB4 generation) mRNA transcripts after treatment with icosabutate. Transcripts for the lipoxygenase genes, ALOX5 and ALOX15, were reduced but constitutive expression levels were low (Figure S1, section B). With respect to PC‐AA content, a significant decrease was also observed in LPCAT2 expression, which is responsible for incorporation of AA into cellular membranes, along with a trend towards lower expression of cPLA2, responsible for AA release. Taken as a whole, the data suggest a downregulation of the AA metabolism by icosabutate at multiple junctures, including decreased AA stores in both PC and DAG stores, decreased HETE concentrations and downregulation of genes regulating eicosanoid synthesis and AA membrane remodelling.

3.5. Icosabutate reduces hepatic oxidative stress and oxidised phospholipids in AMLN ob/ob mice

As highly unsaturated fatty acids are susceptible to peroxidation and can increase hepatic oxidative stress, ³⁰ we also measured hepatic oxidised (GSSG) glutathione and the GSSG/reduced glutathione (GSH) ratio as a marker of oxidative stress and cellular redox status. ³¹ Icosabutate (90 and 135 mpk) significantly decreased hepatic GSSG concentrations (Figure 4G), which in turn was largely responsible for the markedly increased GSH/GSSG ratio (84% increase, P < .01) seen with the highest icosabutate dose (Figure 4H). In conjunction with the increased GSH/GSSH ratio, significant decreases in the hepatic expression of genes regulating enzymatic antioxidants catalase (CAT), and superoxide dismutase 1 and 2 were also observed (Figure S1, section C).

With respect to the functional consequence of increased hepatic oxidative stress, it has recently been demonstrated that hepatic oxidised phospholipids (oxPL) are a causal driver of NASH. ³² We therefore also measured a mixture of the dominant oxPLs (PC 16:0/AA‐OH and LPC 16:0/LA‐OH) and found that icosabutate significantly reduced their concentration by 31% (P < .05) and 46% (P < .001) for the 90 and 135 mpk doses respectively, whereas no effects were seen in response to OCA treatment (Figure 4I).

3.6. Icosabutate primarily _targets highly unsaturated DAG and TAG species in AMLN ob/ob mice

As specific DAG and ceramide species are associated with steatohepatitis ³⁰ and insulin resistance ³¹ in addition to serving as the main source of hepatic AA, ³² we also analysed qualitative differences within the main lipid classes in response to icosabutate (135 mpk shown) or OCA. Whereas OCA primarily reduced TAG and DAG species with 2 to 4 double bonds, icosabutate induced a remarkable reduction in the long and highly unsaturated TAG and DAG species, including AA containing species (Figure 5A and B). Changes in hepatic ceramide species demonstrated a different pattern (Figure 5C), with reductions in response to icosabutate in both shorter and longer chain species, including a 30% reduction in Cer(d18:1/16:0), a species with a deleterious effect on glycemic control. ³¹ Overall the data suggest major differences between icosabutate and OCA with respect to both quantitative and qualitative changes in hepatic lipotoxic lipid species.

3.7. Icosabutate reduces hepatic apoptosis in AMLN ob/ob mice

As a decrease in apoptosis could provide a mechanistic link between the treatment related changes in hepatic lipotoxicity/oxidative stress and the decrease in fibrosis, ¹ we performed a terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay in all groups at study end. Icosabutate reduced apoptotic cell numbers by 41% (P < .01), 29% (P < .05) and 44% (P < .01) vs vehicle in the 45, 90 and 135 mpk dose groups respectively (Figure 6A). In contrast, OCA had no significant effect on apoptotic cell numbers. Representative images of liver cross sections stained with TUNEL are shown in Figure 6B. Overall the data demonstrate that icosabutate effectively decreases apoptosis in AMLN ob/ob mice.

3.8. Icosabutate decreases plasma TAG and total cholesterol but does not affect HDL cholesterol in APOE*3Leiden.CETP mice

The effects of 4 weeks of treatment with either icosabutate or fenofibrate on the main circulating plasma lipids were assessed in APOE*3Leiden.CETP mice. Both icosabutate and fenofibrate significantly decreased plasma TAG (Figure 7A) to a similar extent (−70% for both compounds, P < .001), whereas icosabutate had a more pronounced effect on total cholesterol (−68% and −47% respectively, both P < .001, Figure 7B). Fenofibrate increased HDL cholesterol by 62% (P < .05) whereas icosabutate was without effect (Figure 7C).

3.9. Icosabutate increases VLDL plasma clearance and hepatic uptake of cholesterol and TAG in APOE*3Leiden.CETP mice

To investigate the mechanism/s by which icosabutate lowers plasma lipids, we evaluated very low‐density lipoprotein (VLDL) production and clearance as determinants of plasma VLDL‐TAG levels as compared to fenofibrate. ²⁸ In contrast to icosabutate, fenofibrate increased VLDL‐TAG production compared to controls (+25%) (Figure 7D). Since VLDL‐associated ApoB production and VLDL composition were similar in both groups (data not shown) these data indicate production of larger TAG‐rich VLDL by fenofibrate as reported previously. ²⁸

Compared to controls, the half‐life of glycerol tri[³H]oleate (−40%, P = .008; −46%, P = .005, respectively) (Figure 7E) and [¹⁴C]cholesteryl oleate (−52%, P = .001; −63%, P = .001, respectively) (Figure 7F) were decreased to a similar extent in both icosabutate‐ and fenofibrate‐treated mice. Total uptake of glycerol tri[³H]oleate‐derived lipids was most prominent in liver and skeletal muscle tissue, whereby icosabutate and fenofibrate increased ³H‐activity in the liver compared to vehicle controls (Figure S2A). Uptake of [¹⁴C]cholesteryl oleate by the liver was increased by both treatments compared to controls (Figure S2B). Post‐heparin activity of hepatic lipase (Figure 7G) and lipoprotein lipase (Figure 7H) did not significantly differ between control‐ and icosabutate‐treated animals, whereas fenofibrate increased lipoprotein lipase activity as reported previously. ²⁸ Combined, these data show that the lipid‐lowering effect of icosabutate is mediated through increased hepatic VLDL remnant clearance and may be independent from lipase‐mediated hydrolysis of VLDL‐TAG.

LDL‐R‐mediated hepatic uptake of ApoB‐containing lipoproteins is the most important pathway for removal of these atherogenic particles from the circulation. Both icosabutate and fenofibrate increased hepatic LDL‐R protein expression compared to controls (Figure 7I). These findings indicate that icosabutate increases LDL‐R‐mediated cholesterol uptake in the liver.

3.10. Effect of icosabutate on liver lipid content and faecal bile acid excretion in APOE*3Leiden.CETP mice

To assess if reductions in plasma lipids were associated with hepatic cholesterol/TAG accumulation, decreased cholesterol absorption or increased excretion, hepatic lipid concentrations of cholesterol and TAG along with faecal excretion of bile/neutral sterols were measured. Both treatments decreased hepatic cholesteryl ester content (Figure 7J). Compared to controls, faecal excretion of bile acids was reduced by icosabutate and fenofibrate, whereas neutral sterol contents were unaffected (Figure 7K). These findings negate the possibility that liver accumulation accounts for the decrease in plasma lipids.

3.11. Icosabutate modulates hepatic expression of genes regulating lipid metabolism and inflammation in APOE*3Leiden.CETP mice

A partial overlap between icosabutate and fenofibrate was observed for differentially expressed genes (DEG) (Figure S3A), which could be expected given that fatty acids and their metabolites act as endogenous ligands for PPAR‐α. ⁹ In line with the physiological data, upregulated DEG in the icosabutate group were enriched for pathways involved in lipid metabolism, including fatty acid metabolism and β‐oxidation, and (chole)sterol biosynthetic processes (Figure S3B). Downregulated pathways include complement activation, the AA cascade, innate immune response and acute phase response‐related pathways (Figure S3C). Using computational pathway analysis, Z factor scores were calculated to identify the most dominant transcription factors involved in transcriptional control following icosabutate treatment. Transcription factors that are predicted to be involved in icosabutate‐mediated pathway activation are PPAR‐α and ‐γ, and sterol regulatory element‐binding transcription factor (SREBF)‐1 and −2 and PPAR gamma coactivator (PGC)‐1α (Figure S3D).

3.12. Prolonged low‐dose icosabutate decreases atherosclerotic lesion formation in APOE*3Leiden.CETPmice

Given the marked effect of icosabutate treatment on reduction of plasma cholesterol and TAG, we examined if icosabutate affects aortic root plaque formation in APOE*3Leiden.CETP mice. For 17 weeks animals were treated with vehicle, low‐dose (37.5 mpk for first 4.5 weeks of treatment and 15 mpk for 12.5 weeks) icosabutate, or fenofibrate. This dose regimen achieved a reduction in cholesterol exposure that approximated the 29% reduction in LDL‐C observed in hypercholesterolaemic subjects after treatment with 600 mg/day icosabutate for 28 days. ³³

Representative histological photomicrographs (Figure 8A) demonstrate reduced plaque formation and smaller plaques in the icosabutate‐ and fenofibrate‐treated mice. Reductions in total cholesterol exposure were comparable for the icosabutate and fenofibrate groups and significantly lower than the control group (Figure 8B). Total lesion area was similarly reduced by both icosabutate and fenofibrate (Figure 8C) whereas only icosabutate significantly reduced lesion number (Figure 8D). Both compounds significantly reduced the formation of severe type IV‐V lesions with a concomitant increase in undiseased segments (Figure 8E) albeit these changes were more pronounced in the icosabutate‐treated group. These findings show that low‐dose icosabutate treatment effectively decreases both the development and severity of atherosclerotic lesions.