Nitro fatty acids: novel drug candidates for the co-treatment of atherosclerosis and non-alcoholic fatty liver disease

Nitro-fatty acids (NO₂-FAs) have emerged as a new class of bioactive lipids that mediate metabolic and anti-inflammatory signaling actions. Formed endogenously by the reaction of reactive nitrogen species with unsaturated fatty acids, NO₂-FAs modulate signaling actions mainly via nitro-alkylation of cysteine or histidine residues in transcriptional regulatory proteins. Initial studies identified NO₂-FAs as potent activators of peroxisome proliferator-activated receptors (PPAR), mainly PPARɣ [1]. Subsequently, NO₂-FAs were found to potently inhibit nuclear factor kappa B (NF-κB) signaling [2], and to induce nuclear factor erythroid 2-related factor 2 (Nrf2) signaling [3]. Thus, mediated through post translational modification of key regulatory proteins, NO₂-FAs modulate glucose and lipid metabolism as well as inflammatory and anti-oxidant responses. As these pathways play major roles in the pathogenesis of cardiometabolic diseases, NO₂-FAs have demonstrated protective effects in numerous relevant disease models.

Characterized by imbalanced lipid metabolism, increased oxidative stress and inflammation, atherosclerosis is the underlying cause of most cardiovascular diseases (CVD). While NO₂-FAs are known to reduce vascular inflammation by inhibiting toll-like receptor 4 and NF-κB signaling [4], recent reports have improved our understanding of NO₂-FA metabolism during inflammation and their anti-inflammatory properties. Conjugated linoleic acid (CLA) is a major fatty acid target of nitration reactions. Formation of nitrated CLA (NO₂-CLA) was recently identified in response to acute inflammation both in vitro and in vivo. In turn, NO₂-CLA inhibited NF-κB-dependent gene expression, decreased pro-inflammatory cytokine production and induced Nrf2-regulated anti-oxidant proteins [5*]. The stimulator of interferon genes (STING) has emerged as a critical signaling molecule in immunity and inflammation. NO₂-CLA was found to be formed in response to virus infection and together with nitrated oleic acid (NO₂-OA) was shown to inhibit STING signaling via nitro-alkylation, attenuating the release of type I INF (INF) [6**]. Indeed, the potent anti-inflammatory and anti-oxidant properties of NO₂-FAs were shown by Rudolph et al. [7**] to reduce atherosclerosis in apolipoprotein E-deficient (Apoe^−/−) mice fed an atherogenic diet.

The potent effects of NO₂-FAs were described in the major cell types involved in the pathogenesis of atherosclerosis (Figure 1). In endothelial cells, nitrated linoleic acid (NO₂-LA) and NO₂-OA inhibited lipopolysaccharides (LPS)-induced expression of vascular cell adhesion molecule-1 and monocyte adhesion [2]. This was recently supported by unbiased RNA sequencing, revealing a distinct transcriptome regulated by NO₂-CLA in primary human coronary artery endothelial cells. Modulation of pathways regulating inflammation, oxidative stress, fluid shear stress and atherosclerosis suggested that NO₂-CLA inhibited endothelial dysfunction, a crucial step in atherosclerosis initiation [8]. Macrophage foam cell formation is a hallmark feature of early atherosclerosis and was shown to be attenuated by NO₂-OA which inhibited oxidized low-density lipoprotein (oxLDL)-induced phosphorylation of signal transducer and activator of transcription-1 [7**], increased activity of endogenous anti-oxidants (glutathione and paraoxonase-2) and modulated lipid metabolism in macrophages [9]. Vascular smooth muscle cells are involved in various processes throughout the progression of atherosclerosis. Unbiased transcriptomic profiling revealed that NO₂-CLA regulated critical pathways in human coronary artery smooth muscle cells, including cell proliferation, anti-oxidant response, lipid metabolism and inflammation [10].

Fig. 1. NO₂-FAs for the co-treatment of atherosclerosis and NAFLD: molecular and metabolic mechanisms.

ALT, alanine aminotransferase; AST, aspartate aminotransferase; COL1A, collagen, type I, alpha; CTGF, connective tissue growth factor; GCLC, glutamate-cysteine ligase catalytic subunit; GCLM, glutamate-cysteine ligase regulatory subunit; GPAT2, glycerol-3-phosphate acyltransferase-2; GSH, glutathione; GSR, glutathione reductase; HMOX1, heme oxygenase-1; ICAM1, intercellular adhesion molecule-1; IL-6, interleukin 6; MCP-1, monocyte chemoattractant protein-1; NAFLD, non-alcoholic fatty liver disease; NF-κB, nuclear factor kappa B; NQO1, NAD(P)H quinone dehydrogenase-1; Nrf2, nuclear factor erythroid 2-related factor-2; OxLDL, oxidized low-density lipoprotein; PON2, paraoxonase-2; ROS, reactive oxygen species; SCD-1, stearoyl-CoA desaturase-1; SREBF1, sterol regulatory element-binding transcription factor-1; SREBP1, sterol regulatory element-binding protein-1; STAT-1, signal transducer and activator of transcription-1; TGFB1, transforming growth factor beta-1; TIMP1, TIMP metallopeptidase inhibitor-1; TLR, toll-like receptor; Tumor necrosis factor; VCAM1, vascular cell adhesion molecule-1.

A large body of evidence indicates that atherosclerosis is promoted by non-alcoholic fatty liver disease (NAFLD) which is reaching global epidemic proportions. CVD is the leading cause of death in NAFLD patients, particularly those with non-alcoholic steatohepatitis (NASH) [11]. Recently, we and others identified the therapeutic potential of NO₂-FAs against NAFLD. In mice fed high-fat diet (HFD) for 20 weeks, Khoo et al. [12**] found that treatment with NO₂-OA prevented HFD-induced triglyceride accumulation and mitochondrial dysfunction in the liver. We applied a long-term (24 weeks) murine NASH model induced by high-fructose, high-fat and high-cholesterol diet. Steatosis and fibrosis were confirmed prior to NO₂-OA administration using non-invasive and histological approaches. We found a robust reduction of circulating liver damage markers, hepatic steatosis, lobular inflammation and fibrosis by NO₂-OA. Unbiased transcriptomics uncovered inflammation, fibrogenesis and lipogenesis as major pathways suppressed by NO₂-OA in the liver and a robust inhibition of sterol regulatory element-binding protein-1 (SREBP1) proteolytic activation was noted. Accordingly, NO₂-OA inhibited triglyceride biosynthesis and accumulation in hepatocytes and suppressed fibrogenesis in hepatic stellate cells (Figure 1) [13**]. During these studies, we also tested the effects of NO₂-OA in Apoe^−/− mice fed a Western diet and reported reduced hepatic steatosis [13**]. Herein, we show that treatment with NO₂-OA simultaneously reduced atherosclerosis in Apoe^−/− mice (Figure 2).

Fig. 2. NO₂-OA prevents Western diet-induced atherosclerosis in Apoe^−/− mice.

Male Apoe^−/− mice were fed standard chow diet or Western diet (Envigo TD.88137, 42% fat and 0.2% cholesterol) for 8 weeks and treated with 8 mg/kg/day of NO₂-OA via subcutaneously implanted osmotic mini-pumps. Mice fed chow diet and administrated vehicle (polyethylene glycol) as well as mice fed Western diet and administrated vehicle or 8 mg/kg/d of non-nitrated OA were served as controls. Oil Red O staining was used for en face analysis of atherosclerotic lesions in the aortic tree. Representative aortas from each experimental group are shown. Statistical differences were compared by one-way ANOVA followed by Bonferroni post-hoc test (n=8 mice per group).

Beyond the above studies applying various experimental models, NO₂-FAs are currently evaluated in several clinical trials. Particularly, CXA-10, a specific regioisomer of NO₂-OA (10-nitro-octadec-9-enoic acid) underwent pharmaceutical development as a drug candidate for the treatment of inflammatory and fibrotic diseases using oral and intravenous formulations. As phase II studies are currently ongoing in patients with focal segmental glomerulosclerosis and pulmonary arterial hypertension, a recent phase I study reported reductions in serum biomarkers of inflammation (monocyte chemoattractant protein-1, MCP1) and metabolic dysfunction (triglycerides) following oral administration of CXA-10 to obese subjects [14**]. Considering the supportive findings from preclinical and clinical studies, the lack of approved drugs for NAFLD and its global burden together with the challenges of simultaneously treating CVD and NAFLD [15], the co-treatment of atherosclerosis and NAFLD using NO₂-FAs warrants further clinical evaluation.