Abstract

Background

   Nonalcoholic fatty liver disease (NAFLD) and resulting nonalcoholic
   steatohepatitis (NASH) are reaching global epidemic proportions. Lack
   of non-invasive diagnostic tools and effective therapies constitute two
   of the major hurdles for a bona fide treatment and a reversal of NASH
   progression and/or regression of the disease. Nitro-oleic acid
   (OA-NO[2]) has been proven effective in multiple experimental models of
   inflammation and fibrosis. Thus, the potential benefit of in vivo
   administration of OA-NO[2] to treat advanced NAFLD was tested herein in
   a model of long-term NASH diet-induced liver damage.

Methods

   Non-invasive imaging (e.g. photoacustic-ultrasound (PA-US)) was pursued
   to establish advanced experimental model of NASH in mice in which both
   steatosis and fibrosis were diagnosed prior experimental therapy with
   OA-NO[2]. Experimental controls included equimolar amounts of the
   non-nitrated oleic acid (OA). CLAMS and NMR-based analysis was used for
   energy metabolism.

Findings

   CLAMS and NMR-based analysis demonstrates that OA-NO[2] improves body
   composition and energy metabolism and inhibits hepatic triglyceride
   (TG) accumulation. Photoacoustic-ultrasound imaging revealed a robust
   inhibition of liver steatosis and fibrosis by OA-NO[2]. RNA-sequencing
   analysis uncovered inflammation and fibrosis as major pathways
   suppressed by OA-NO[2] administration, as well as regulation of
   lipogenesis and lipolysis pathways, with a robust inhibition of SREBP1
   proteolytic activation and subsequent lipogenesis gene expression by
   OA-NO[2]. These results were further supported by histological analysis
   and quantification of lipid accumulation, lobular inflammation (F4/80
   staining) and fibrosis (collagen deposition, αSMA staining) as well as
   established parameters of liver damage (ALT). In vitro studies indicate
   that OA-NO[2] inhibits TG biosynthesis and accumulation in hepatocytes
   and inhibits fibrogenesis in human stellate cells.

Interpretation

   OA-NO[2] improve steatohepatitis and fibrosis and may constitute an
   effective therapeutic approach against advanced NAFLD that warrants
   further clinical evaluation.

   Keywords: Nitro-fatty acids; Non-alcoholic fatty liver disease;
   Non-alcoholic Steatohepatitis; Non-invasive liver imaging, liver
   fibrosis
     __________________________________________________________________

Research in context.

Evidence before this study

   With nonalcoholic fatty liver disease (NAFLD) and resulting
   nonalcoholic steatohepatitis (NASH) becoming a growing concern and a
   major health care issue, efficient therapies to date are
   underdeveloped. Previous progress in preclinical development of
   nitro-fatty acids as anti-inflammatory lipid mediators have uncovered
   benefits on experimental models of metabolic and aligned cardiovascular
   diseases. However, the therapeutic potential of nitro-fatty acids
   against NAFLD has not been investigated.

Added value of this study

   This study demonstrates that nitro-oleic acid (OA-NO[2]), a
   prototypical component of the nitro-fatty acids, is protective against
   NAFLD in two independent experimental settings: 1) western diet-induced
   progressive steatosis (apoE knockout mice) and 2) established NASH by
   long-term administration of high-cholesterol, high fructose diet in
   wild-type mice (NASH model). A non-invasive imaging approach applied to
   a NASH model of liver steatosis and fibrosis was further pursued with
   two aims: 1) to mimic an ideal scenario for NASH diagnosis (avoiding
   liver biopsy); and 2) to evaluate the putative therapeutic benefits of
   nitro-fatty acids against established NASH. This study demonstrates
   that OA-NO[2] reduces clinically-relevant markers of liver damage, and
   protects against lipid accumulation, lobular inflammation and fibrosis
   aligned with an overall improvement of metabolism. Both in vivo and in
   vitro studies demonstrate that OA-NO[2] inhibits triglyceride
   biosynthesis and accumulation in hepatocytes while inhibiting
   profibrotic activation of human stellate cells and reducing collagen
   deposition in the liver.

Implications of all the available evidence

   Current progress in preclinical development of OA-NO[2] has translated
   into clinical therapeutics in ongoing phase II clinical trials. Yet,
   our findings expand current knowledge and uncovers OA-NO[2] as readily
   viable candidates for therapy against NAFLD. While some ongoing trials
   include obese patients that may provide translational validation of the
   putative therapeutic benefits of OA-NO[2] against NAFLD, clinical
   trials specifically designed to test the therapeutic value of OA-NO[2]
   derivatives for NASH will be critical to pursue.

   Alt-text: Unlabelled Box

1. Introduction

   Nonalcoholic fatty liver disease (NAFLD) is a spectrum of liver
   disorders characterized by hepatic fat accumulation, inflammation, and
   hepatocyte injury [[55]1,[56]2]. In concert with the increased
   prevalence of obesity due to excess caloric intake and a sedentary
   lifestyle, the prevalence of NAFLD is increasing worldwide, affecting
   an estimated 24% of the population [[57]3,[58]4]. NAFLD is associated
   with increased mortality related to cardiovascular disease (CVD),
   malignancy and liver disease [[59]3,[60]5]. In up to 40% of
   individuals, hepatic steatosis progresses to nonalcoholic
   steatohepatitis (NASH), further characterized by inflammatory
   infiltrate and hepatocyte injury. A subset of patients develops
   progressive collagen deposition (fibrosis) [[61]6]. To date, no
   therapies are available for NASH [[62]6]. Thus identification of novel
   therapeutic targets against hepatic steatosis, liver inflammation and
   fibrosis is a major clinical need [[63]7].

   Nitroalkene derivatives of unsaturated fatty acids (NO[2]-FAs) have
   emerged as potent anti-inflammatory and anti-fibrotic signaling
   mediators [[64]8,[65]9]. NO[2]-FAs are generated during inflammation
   and digestion through non-enzymatic reactions of unsaturated fatty
   acids with nitrogen dioxide (^.NO[2]), yielding an array of
   electrophilic NO[2]-FAs with unique biochemical and signaling
   properties [[66]10,[67]11]. Nitro-oleic acid (OA-NO[2]), exerts
   protective roles in numerous experimental models of inflammation,
   imbalanced lipid metabolism and fibrosis [[68]9]. These include
   endotoxin-induced vascular inflammation and multi-organ injury
   [[69]12,[70]13], colitis [[71]14], atherosclerosis [[72]15], systemic
   and pulmonary arterial hypertension (PAH) [[73]16,[74]17], atrial
   fibrillation and myocardial fibrosis [[75]18,[76]19]. The safety and
   pharmacokinetics of OA-NO[2] have been clinically examined in four
   successfully completed phase I trials ([77]NCT02127190,
   [78]NCT02248051, [79]NCT02460146, [80]NCT02313064) and its therapeutic
   potential currently evaluated in phase II clinical trials for the
   treatment of focal segmental glomerulosclerosis (FSGS), PAH and asthma
   [[81]9].

   Considering the unique pathogenesis of NAFLD involving imbalanced
   hepatic lipid metabolism, inflammation and fibrosis [[82]1,[83]2] along
   with the well-established anti-inflammatory and anti-fibrotic
   properties of OA-NO[2] [[84][7], [85][8], [86][9], [87][10], [88][11],
   [89][12], [90][13], [91][14], [92][15], [93][16], [94][17]] it is
   plausible to suggest that OA-NO[2] will have a protective effect
   against NAFLD. Herein, we provide evidence for the protective role of
   OA-NO[2] against NASH, using non-invasive imaging for NASH diagnosis
   coupled with histological, molecular and biochemical approaches. Thus,
   our study provides functional evidence to uncover the putative
   therapeutic assessment of OA-NO[2] in steatohepatitis and fibrosis.

2. Materials and methods

2.1. Animal procedures

   Eight weeks-old male C57BL/6J (RRID: IMSR_JAX:000664) and apoE^−/−
   (RRID: IMSR_JAX:002052) mice were fed standard chow diet (CD),
   Western-diet (WD, Supplementary Tables 1 and 2) or NASH-diet rich in
   saturated fat, trans-fat, fructose and cholesterol (Supplementary
   Tables 3 and 4) for 24 weeks. OA-NO[2] was administered via osmotic
   minipump implantation as described in the Supplementary Materials and
   Methods.

2.2. Non-invasive in vivo imaging for dual analysis of hepatic steatosis and
fibrosis

   Hepatic lipid and collagen contents were quantitatively determined in
   vivo in a subgroup of mice by photoacoustic imaging using a
   photoacoustic-ultrasound (PA-US) dual modality system [[95]20]
   described in the Supplementary Materials and Methods. During the
   procedure, mice were anesthetized with ketamine (100 mg/kg) and
   xylazine (10 mg/kg) injection.

2.3. Liver steatosis, lobular inflammation and fibrosis scoring

   H&E and Sirius red staining were used to score liver steatosis, lobular
   inflammation and fibrosis as previously described [[96]21] and
   elaborated in the Supplementary Materials and Methods.

2.4. Statistical analysis

   Statistical analyses were performed using SPSS 24.0 software (SPSS Inc.
   IBM). Unless indicated otherwise, values are presented as box-plots and
   whiskers or means ± SEM of at least three independent observations. The
   number of animals or experiments used for each study is specified for
   each figure legend. One-way analysis of variance (ANOVA) followed by
   Bonferroni post hoc test was used for data analysis. Differences were
   considered statistically significant at p < .05.

2.5. Sample-size estimation

   The number of mice used for the long-term NASH study was determined by
   sample-size analysis and calculated based on our initial studies with
   apoE^−/− mice (Supplementary Fig. 1), using hepatic TG content as the
   primary endpoint measure. Using WinPepi statistical software, assuming
   α error rate of 0.05 and β error rate of 0.20, the number of mice for
   the NASH study was n = 10 per group.

2.6. Blinding

   The following measurements were conducted by technicians blinded to the
   mouse experimental groups: Liver imaging, pathology, CLAMS, body
   composition analysis, plasma lipids, and plasma cytokines.

2.7. RNA-sequencing biorepository

   The RNA-sequencing data discussed in this publication have been
   deposited in NCBI's Gene Expression Omnibus and are accessible through
   GEO Series accession number [97]GSE126204
   [98]www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE126204

2.8. Ethics statement

   All animal procedures were approved by the Institutional Animal Care &
   Use Committee of the University of Michigan (PRO00006176) and performed
   in accordance with the institutional guidelines.

3. Results

3.1. OA-NO[2] protects against liver steatosis and fibrosis during NASH
development

   The benefits of OA-NO[2] in pathologies related with imbalanced lipid
   metabolism were originally observed in hyperlipidemic apoE^−/− mice in
   which administration of OA-NO[2] during western-diet (WD) feeding
   reduced atherosclerosis [[99]15]. However, whether OA-NO[2] can
   attenuate WD-induced lipid accumulation in the liver is unknown. In
   initial studies, treatment with OA-NO[2], but not with non-nitrated OA,
   prevented early steatosis in apoE^−/− mice (Supplementary Fig. 1a-1c),
   attenuated WD-induced hepatic overexpression of key lipogenic genes
   (e.g. SREBF1, SCD1, Supplementary Fig. 1d) and decreased plasma TG
   without significant effects on plasma TC, LDL or HDL (Supplementary
   Fig. 1e-h). To determine the therapeutic use of OA-NO[2] against NASH,
   we devised an experimental approach intended to model more advanced
   stages of NAFLD, a realistic scenario characterized by established
   coexistence of steatosis and fibrosis ([100]Fig. 1a). To monitor NASH
   progression, a novel imaging technique was used that provides an
   accurate simultaneous measurement of both hepatic steatosis and
   fibrosis in a non-invasive manner. C57BL/6 mice were fed with a NASH
   diet for 12 weeks (Supplemental Table 3 and 4) and liver pathology was
   evaluated using high resolution physio-chemical analysis (PCA)
   [[101]22]. A subset of the animals was sacrificed to confirm
   established liver steatosis and early fibrosis by histology. Liver PCA
   revealed enhanced fingerprints in optical wavelengths of 1220 nm and
   1370 nm, which identify hepatic lipid and collagen content,
   respectively ([102]Fig. 1b-c). Histological analysis based on H&E and
   Oil Red O staining confirmed lipid accumulation and hepatocyte
   ballooning (Supplementary Fig. 2a-b) while Sirius red staining
   confirmed collagen deposition and early fibrosis (Supplementary Fig.
   2c).

Fig. 1.

   [103]Fig. 1
   [104]Open in a new tab

   Non-invasive diagnosis reveals OA-NO[2] protection against NASH-diet
   induced hepatic steatosis and fibrosis. (a) Experimental design:
   Steatohepatitis was induced in C57BL/6 mice by a NASH diet, rich in
   saturated fat, trans fat, fructose and cholesterol for 12 weeks. After
   12 weeks, high-resolution physio-chemical analysis ultrasound (PCA-US),
   confirmed coexistence of lipid steatosis and early fibrosis. Then,
   osmotic minipumps were implanted subcutaneously to deliver PEG, OA or
   OA-NO[2] (5 mg/kg/d) for additional 12 weeks under chow diet (CD) or
   NASH-diet feeding (4 groups, n = 10 per group). Established liver
   damage was confirmed in a subpopulation (n = 3) analyzed after 12 weeks
   using (b) conventional ultrasound (US) combined with high-resolution
   PCA high-resolution at 1220 nm optical wavelength to detect hepatic
   lipids, and (c) at 1370 nm optical wavelength to quantify hepatic
   collagen content. Two weeks before terminal analysis of liver pathology
   PA-US was used to quantitatively analyze lipid content and total
   collagen content in the NASH-diet model. High-resolution PCA
   demonstrated a marked reduction of total hepatic lipid (d) and collagen
   content (e) upon OA-NO[2] treatment compared to non-nitrated OA.
   Quantitative analysis of PA absorption from each experimental group at
   1220 nm (f) and 1370 nm fingerprint (g). Size bars = 5 mm Data is
   plotted as box and whiskers from minimum to maximum values showing all
   points. *p < .05, **p < .01, ***p < .001 vs. CD; ^^p < .05, vs. NASH
   OA. n = 8.

   After confirmatory concurrence of hepatic steatosis and fibrosis,
   animals were randomized to receive OA-NO[2], OA as non-nitrated fatty
   acid control and PEG, which served as vehicle, via osmotic minipumps
   implantation for additional 12 weeks under ad libitum NASH-diet feeding
   ([105]Fig. 1a). Two weeks before terminal analysis, non-invasive
   photoacoustic ultrasound (PA-US) based-imaging was further pursued in
   all experimental groups to quantitatively determine whether OA-NO[2]
   treatment was effective against progressive NASH. Liver pathology
   evaluated using high resolution PCA revealed a progressively enhanced
   fingerprints of hepatic lipid content (1220 nm optical wavelength,
   [106]Fig. 1d) and collagen content (1370 nm optical wavelength,
   [107]Fig. 1e) in NASH diet-fed mice treated with either PEG or OA
   compared to CD-fed mice. Treatment with OA-NO[2] markedly suppressed
   both NASH diet-induced hepatic lipid accumulation and collagen
   deposition, quantified by the lower magnitude of the PA signals at
   1220 nm ([108]Fig. 1f) and 1370 nm ([109]Fig. 1g), respectively.

3.2. OA-NO[2] improves body composition and promotes a metabolic phenotype in
NASH-diet fed mice similar to CD feeding

   Gross appearance of the peritoneal cavities at the experimental
   endpoint (24 weeks) revealed substantial hepatic steatosis with
   enlarged visceral fat pads in NASH-diet fed mice administrated with PEG
   or OA, which were attenuated by OA-NO[2] ([110]Fig. 2a). Body-weight
   measurement, showed a trend towards reduced body weight gain upon
   OA-NO[2] administration, reaching statistical significance compared
   with NASH PEG group but not with OA (p < .05, Supplementary Fig. 3a).
   Yet, NMR-based analysis of body composition indicated that body fat was
   significantly reduced by OA-NO[2] along with a significant increase in
   lean body mass ([111]Fig. 2b-c).

Fig. 2.

   [112]Fig. 2
   [113]Open in a new tab

   OA-NO[2] improves body composition and increases respiratory quotient
   in NASH-diet induced steatohepatitis. (a) Gross appearance of the
   peritoneal cavity at the experimental end-point (24 weeks) depicting
   apparent reduction of liver steatosis upon treatment with OA-NO[2]. One
   week before end-point analysis, NMR-based body composition analysis was
   conducted (n = 8) revealing a significant reduction of % body fat (b)
   and subsequent increase in % lean body mass (c), upon OA-NO[2]
   treatment. No alteration in body composition was observed in mice
   treated with equimolar amounts of non-nitrated OA. Quantitative data is
   plotted as box and whiskers from minimum to maximum values showing all
   points. (d) OA-NO[2] increased the respiratory exchange ratio (RER)
   calculated as the ratio of CO[2] generation vs. oxygen consumption
   (VCO[2]/VO[2]) assessed by CLAMS. Data are shown over a 24 h dark/light
   period cycle (n = 8). (e) Quantitative analysis of RER in each
   experimental group shown as box and whiskers from minimum to maximum
   values. Data is shown as mean ± SEM. *p < .05, **p < .01, ***p < .001
   vs CD PEG; ^#p < .05, ^##p < .01, ^###p < .001 vs. NASH PEG ^^p < .05
   vs. NASH OA.

   The metabolic response to obesity involves a shift towards greater
   lipid versus lower carbohydrate utilization, reflecting a reduction in
   respiratory quotient (respiratory exchange ratio-RER)
   [[114]23,[115]24]. Accordingly, CLAMS analysis indicated a pronounced
   reduction of RER in response to NASH-diet, both in the OA or PEG
   vehicle-treated controls ([116]Fig. 2d-e), reflecting an increased fat
   oxidation (Supplementary Fig. 4a), and decreased glucose oxidation
   (Supplementary Fig. 4b) in these experimental groups. OA-NO[2]
   treatment, however, partially reversed this trend towards a reduced fat
   oxidation and increased glucose oxidation, resulting in a significant
   elevation of the respiratory quotient ([117]Fig. 2d-e). No significant
   differences in energy expenditure, or ambulatory activity
   (Supplementary Fig. 4c-d) were observed between the groups.

3.3. OA-NO[2] protects against steatohepatitis, hepatomegaly and liver damage
in NASH

   Chronic NASH-diet feeding for a total of 24 weeks resulted in a
   significant increase in body weight irrespectively of intervention with
   PEG or OA (p < .001 or p < .05, respectively) compared to CD, with a
   similar trend in mice treated with OA-NO[2] (p = .113, Supplementary
   fig. 3a). Livers were drastically enlarged in the PEG- and OA-treated
   group (3.28 ± 0.12 g, 3.06 ± 0.14, respectively, p < .001, [118]Fig. 3a
   and Supplementary Fig. 3b) but not in the OA-NO[2] group, which
   exhibited significantly reduced liver to body weight ratios ([119]Fig.
   3b).

Fig. 3.

   [120]Fig. 3
   [121]Open in a new tab

   OA-NO[2] prevents NASH diet-induced hepatomegaly and liver damage, and
   improves NAFLD activity scores. (a) Gross liver morphology, H&E and Oil
   Red O histology from each experimental group. H&E histology was used
   for quantitative NAFLD scoring as described and summarized in
   [122]Table 1. (b) Quantitative analysis of hepatomegaly was determined
   as of liver to body weight ratio. (c) Fasting plasma analysis of
   alanine aminotransferase (ALT) and (d), aspartate aminotransferase
   (AST). (e) Hepatic triglyceride mass and (f) cholesterol mass from each
   experimental group. Quantitative data is plotted as box and whiskers
   from minimum to maximum values showing all points. *p < .05, **p < .01,
   ***p < .001 vs CD/PEG; ^#p < .05, ^##p < .01, ^###p < .001 vs. NASH PEG
   ^^p < .05, ^^^p < .01, ^^^^p < .001, vs. NASH OA. (For interpretation
   of the references to colour in this figure legend, the reader is