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. Author manuscript; available in PMC: 2022 Oct 1.
Published in final edited form as: Cardiovasc Drugs Ther. 2020 Jul 15;35(5):939–951. doi: 10.1007/s10557-020-07031-8

Suppression of Vascular Macrophage Activation by Nitro-Oleic Acid and its Implication for Abdominal Aortic Aneurysm Therapy.

Yang Zhao 1,2,#, Ziyi Chang 1,3,#, Guizhen Zhao 1, Haocheng Lu 1, Wenhao Xiong 4, Wenying Liang 1, Huilun Wang 1, Luis Villacorta 1, Minerva T Garcia-Barrio 1, Tianqing Zhu 1, Yanhong Guo 1, Yanbo Fan 1,5, Lin Chang 1, Francisco J Schopfer 6, Bruce A Freeman 6, Jifeng Zhang 1,*, Y Eugene Chen 1,2,*
PMCID: PMC7855321  NIHMSID: NIHMS1614766  PMID: 32671602

Abstract

Purpose

Abdominal aortic aneurysm (AAA) is one of the leading causes of death in the developed world and is currently undertreated due to the complicated nature of the disease. Herein, we aimed to address the therapeutic potential of a novel class of pleiotropic mediators, specifically a new drug candidate, nitro-oleic acid (NO2-OA), on AAA, in a well-characterized murine AAA model.

Methods

We generated AAA using a mouse model combining AAV.PCSK9-D377Y induced hypercholesterolemia with Angiotensin II given by chronic infusion. Vehicle control (PEG-400), oleic acid (OA), or NO2-OA were subcutaneously delivered to mice using osmotic minipump. We characterized the effects of NO2-OA on pathophysiological responses and dissected the underlying molecular mechanisms through various in vitro and ex vivo strategies.

Results

Subcutaneous administration of NO2-OA significantly decreased the AAA incidence (8/28 mice) and supra-renal aorta diameters as compared to mice infused with either PEG-400 (13/19, p=0.0117) or OA (16/23, p=0.0078). In parallel, the infusion of NO2-OA in the AAA model drastically decreased extracellular matrix degradation, inflammatory cytokine levels, and leucocyte/macrophage infiltration in the vasculature. Administration of NO2-OA reduced inflammation, cytokine secretion, and cell migration triggered by various biological stimuli in primary and macrophage cell lines partially through activation of the peroxisome proliferator-activated receptor-gamma (PPARγ). Moreover, the protective effect of NO2-OA relies on the inhibition of macrophage prostaglandin E2 (PGE2)-induced PGE2 receptor 4 (EP4) cAMP signaling, known to participate in the development of AAA.

Conclusion

Administration of NO2-OA protects against AAA formation and multifactorial macrophage activation. With NO2-OA currently undergoing FDA approved Phase II clinical trials, these findings may expedite the use of this nitro-fatty acid for AAA therapy.

Keywords: Aortic Disease, Abdominal Aortic Aneurysm, Nitro-fatty acid, Macrophage

Introduction

Characterized by the enlargement of the abdominal aorta over its standard size (≥ 30mm or 1.5 times), abdominal aortic aneurysm (AAA) is a primary medical concern owing to its wide prevalence, high mortality rate and lack of effective treatment [1, 2]. Although the success achieved in reducing the AAA-related mortality rate through preventive screening in high-risk men between 65 to 75 years, open and endovascular surgeries are still the only treatments available, with just a small proportion of AAA patients eligible for surgical repair [3, 4]. Besides, patients undergoing either surgery are not risk-free. While open repair is associated with a relatively high risk of perioperative mortality, the widely used endovascular repair nowadays also increases the likelihood of postsurgical leaking, leaving an urgent need for alternative therapeutic strategies [4, 5]. Although vascular inflammation, macrophage infiltration, oxidative stress, and extracellular matrix degradation are widely accepted as the pathological features of AAA, their direct causative roles and contribution to progression and to the late stage of the disease are not well-defined [6–8]. While several pharmacologic agents targeting certain individual factors appeared to be promising in preclinical studies, leverage into beneficial outcomes in clinical settings of AAA is not evident to date, likely due to the complex pathophysiological nature of AAA [9].

Nitro-fatty acids (NO2-FAs), represent a convergence between unsaturated fatty acid, nitrogen oxide and electrophile-mediated signaling, acting via pleiotropic mechanisms as inflammatory and metabolic regulators in model systems and humans [10]. Nanomolar concentrations of NO2-FAs can form endogenously in response to nitric oxide- and nitrite-derived nitrogen dioxide generated via local inflammatory and oxidative reactions, as well as in the gastrointestinal tract where low pH favors fatty acid nitration [10–13]. While NO2-FAs participate in reversible Michael addition reactions with cellular nucleophiles, most biological activity is mediated by the post-translational modification of critical cysteines found in regulatory proteins [10, 11]. For instance, NO2-FAs activate PPARγ, inhibit p65-dependent nuclear factor-kappa B (NF-kB) activation, induce the heat shock response, and promote nuclear factor erythroid 2-related factor 2 (Nrf2) dependent anti-oxidative effect [14–18]. Additionally, the pharmacodynamics/pharmacokinetic characteristics of a Phase 1 human study of CXA-10 (10-nitro-oleic acid) showed that CXA-10 is safe within a therapeutic window (25–450mg/day) and attenuated systemic inflammation in human volunteers. This was reflected by decreased circulating monocyte chemoattractant protein 1 (MCP1) and interleukin 6 (IL6) levels [19]. In parallel, in vivo studies administering NO2-OA via either oral or non-oral routes in various animal models, demonstrated the protective effect of NO2-FAs in multiple disease models including atherosclerosis, hypertension, vascular inflammation, cardiac ischemia/reperfusion injury, kidney nephropathy and non-alcoholic fatty liver disease [20–27].

The transport mechanism of NO2-FAs provides a distinct and specific distribution to cells and organs, defining target tissues and cell signaling events. For instance, NO2-FAs in the systemic circulation are esterified to triglycerides (TG). They can be taken up, after hydrolysis, by circulating monocytes, vascular endothelium and macrophages (e.g., through CD36, FABPs) to exert intracellular signaling activities [10, 28–30]. In this regard, a monocyte-vascular-macrophage axis may play a predominant role in the NO2-FA-mediated beneficial vascular effects. This is supported by the observation that the administration of CXA-10 in humans induces Nrf2-regulated genes and heat shock response in peripheral blood mononuclear cells [19, 29]. The fact that NO2-OA decreases differentiation of monocytes to macrophages induced by colony-stimulating factors, inhibits LPS induced leukocyte adhesion to the vascular endothelium, and prevents pro-inflammatory macrophage polarization, further point to the importance of NO2-FAs in regulating monocyte/macrophage biology [31–33].

Herein, we reveal the protective effects of NO2-OA in a mouse model of AAA induced by Angiotensin II (AngII) and hypercholesterolemia and identified significantly decreased leukocyte infiltration into the vascular wall. Using oxidized low-density lipoprotein (oxLDL) and AngII, we recapitulated the anti-inflammatory function of NO2-OA and showed that it could be partially accounted for by enhanced PPARγ gamma signaling and decreased p65-dependent NF-κB activity in cultured RAW264.7 cells and primary bone marrow-derived macrophages (BMDMs). Additionally, we report the biased-regulation of NO2-OA on macrophage prostaglandin E2 (PGE2)-induced activation of the prostaglandin E2 receptor 4 (EP4), a pathway previously associated with human aneurysm development [34, 35].

Materials and Methods

Antibodies and reagents

Antibodies against p65 (#6956), Lamin A/C (#4777), GAPDH (#5174), β-actin (#4970, and #3700) were purchased from Cell Signaling Technology (CST, Danvers, MA). Fluor 488 and 594 conjugated secondary antibodies were from Jackson ImmunoResearch (West Grove, PA). For cell experiments, Ang II (#17150), PGE2 (#14010), ONO-AE3–208 (#14522), GW9662 (#70785) and oleic acid (#90260) were purchased from Cayman Chemical (Ann Arbor, MI). nLDL (#12–16-120412-TC) and oxLDL (#12–16120412-OX) were from Athens Research & Technology (Athens, GA). siPPARγ (D-040712–01-0002), siPtger4 pool (M-048700–01-0005) and siControl pool2 (D-001206–14-05) were from Dharmacon (Lafayette, CO). RNAiMAX (#13778150) and Lipofectamine 2000 (#11668) were from Invitrogen (Waltham, MA). Human N-HA tagged FEM1A (#HG21487-NY) was purchased from Sino Biological (Beijing, China). pCMV4-p105 was from Addgene (plasmid #23288, Watertown, MA). NO2-OA was synthesized, as previously described [36].

Cell culture and isolation of BMDMs

Murine RAW 264.7 cell line, 293T/17 cell line and Primary Umbilical Vein Endothelial Cells (PCS-100–010, HUVECs) were purchased from ATCC (Manassas, VA) and cultured in DMEM (Gibco) with 10% FBS (Thermo Fisher Scientific) or M199 (Gibco) supplemented with 20% FBS for HUVECs. BMDMs isolated from 12 to 16-week-old C57BL/6 wild type mice were cultured in bone marrow macrophage differentiation media. The differentiation medium was prepared by adding L-cell conditioned media (30%), non-essential amino acids (#11140–050, Gibco), sodium pyruvate (1 mM, #11360–070, Gibco), 2-Mercaptoethanol (55 µM) and 10% FBS into Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco) [37].

Animal procedures

Ten weeks old male C57BL/6J mice were purchased from The Jackson Laboratory. The murine AAA model induced by AngII/PCSK9 gain-of-function mutation is well-established [38]. After acclimation for 1 week, mice were fed a western diet (TD.88137, Envigo) and intraperitoneally injected with 2×1011 genomic copies of AAV-PCSK9.D377Y (AAV serotype 8, Penn Vector Core). Two weeks later, each mouse was subcutaneously implanted with two osmotic pumps (Model #2004, Alzet). One was loaded with AngII (#H-1706, Bachem), releasing at a rate of 1,500 ng/kg/min, while the other one was loaded with PEG-400, OA or NO2-OA at a delivery rate of 5 mg/kg/day for OA or NO2-OA [39, 25, 21, 32]. Systolic blood pressure was measured using BP-2000 system. After 4-weeks infusion, mice were euthanized and the maximal outer diameter of the suprarenal aorta was assessed with a digital caliper by a third person blinded to the treatment. Suprarenal aortas with a maximal diameter ≥ 50% larger than standard size (≥ 1.2 mm) were considered as abdominal aortic aneurysms. Mice dead within the first week of pump implantation or with a serum total cholesterol level less than 250mg/dL were excluded from the study. All animal procedures were performed following the protocols approved by the Institutional Animal Care & Use Committee (IACUC) at the University of Michigan.

Total cholesterol (TC) and triglyceride measurement

Serum total TC and TG levels were measured by enzymatic reaction (Wako Diagnostics, Osaka, Japan).

Histology staining

H&E and Verhoeff Van Gieson staining of paraffin or frozen sections of mouse suprarenal abdominal aorta were performed by the In-Vivo Animal Core at the University of Michigan. Elastin degradation grade was calculated as we previously reported [40].

RNA extraction and quantification

Mice suprarenal aortas were collected after the measurement of their diameter. Human aortic aneurysm samples were obtained from the Department of Cardiac Surgery at the University of Michigan (Supplementary Table S1). Samples from humans and mice were snap-frozen in liquid nitrogen for later processing. RNA from tissue was extracted using TRIzol (#15596018, Invitrogen). RNA from cells was isolated with the RNeasy Mini Kit (#74106, QIAGEN). SuperScript™ III First-Strand Synthesis System (#18080051, Invitrogen) was used to reverse-transcribe mRNA into cDNA. Gene expression was quantified in triplicates by quantitative real-time PCR (qPCR) using IQ SYBR Green Supermix (#1708882, Bio-Rad) with the indicated gene primers (Supplementary Table S2).

Enzyme-linked immunosorbent assay (ELISA)

MCP1, TNFα and IL6 levels in mouse plasma were measured by ELISA performed by the Cancer Center Immunology Core at the University of Michigan.

Fluorescence-activated cell sorting (FACS)

The suprarenal aorta region from euthanized mice were completely perfused with PBS containing 2% of heparin. Tissues were digested using a cocktail of 450 U/mL collagenase type I (#17100–017, Gibco), 125 U/mL collagenase type XI (#C7657, Sigma-Aldrich), 60 U/mL hyaluronidase type I-s (#H3606, Sigma-Aldrich,) and 60 U/mL DNase-I (#10104159001, Roche) at 37°C for 90 minutes. Single cells filtered through a 70 µm cell strainer were blocked by Fc block (#14–0161-85, eBioscience) for 5 minutes on ice. After blocking, cells were incubated with fluorochrome-conjugated antibodies against CD45 (#48–0451, eBioscience), CD64 (#558455, BD), and CD11c (#47–0114, eBioscience) for 30 minutes on ice, washed, fixed with 2% PFA, and subjected to FACS analysis by the Flow Cytometry Core at the University of Michigan. Data were analyzed using FlowJo v10.6.1.

Boyden chamber transwell migration assay

HUVECs were seeded in the lower chambers of the Transwells (#CLS3464, Sigma-Aldrich) and cultured up to 80% confluence before treatment with or without AngII (100 nM) for 6 hours in OptiMEM I reduced serum medium (#31985–070, Gibco). RAW 264.7 cells starved with OptiMEM I overnight were seeded (2×105 per well) in the upper chambers of the transwells and treated with vehicle (ethanol), OA (2.5 μM) or NO2-OA (2.5 μM) for additional 12 hours. Cells on the upper chamber were then removed by gently scraping with a cotton swab, and the membranes were fixed in 4% PFA for 15 min and stained with 0.1% crystal violet for 15 min at room temperature. Images were acquired by AMG Evos XI Core Imaging System. Quantification of the positively stained area was performed using Image J v1.52s.

Nuclear/cytoplasm protein extraction, co-immunoprecipitation and immunoblotting

NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (#78835, ThermoFisher) were used for nuclear and cytoplasmic protein extraction. For Co-IP, cells were lysed using Cell Lysis Buffer (#9803, CST) supplemented with PMSF (#8553, CST), immunoprecipitated using Protein G Magnetic Beads (#7024, CST) and antibodies against HA (#AE008, ABclonal) or p50/p105(#13586, CST) following the manufacture’s protocol. For immunoblot, protein extracts were resolved in SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were blocked by 5% BSA and incubated with the primary antibody at 4°C overnight followed by incubation with fluorescence conjugated secondary antibody for 1h at room temperature. Band intensity was collected with the Odyssey® CLx Imaging System and quantified using Image Studio™ Lite.

EP4 receptor activity SEAP assay and cAMP Homogeneous Time-Resolved Fluorescence (HTRF) assay

The effect of NO2-OA on PGE2 induced EP4 activation was assessed using EP4 Receptor (human) Reporter Assay Kit (#601390, Cayman) in 293T/17 cells. Cells at 80% density were incubated on plates coated with transfection complex containing human EP4 receptors and a cAMP response element-regulated secreted alkaline phosphatase (SEAP) reporter. Activation of EP4 was quantified by adding a luminance-based alkaline phosphatase substrate and read in a chemiluminescence plate reader relative to the kit’s standards. EP4 Inhibition assay was performed using cAMP-Gs HiRange kit (#62AM6PEB, Cisbio) following the manufacture’s protocol and measured by Neo2 plate reader (BioTek, Winooski, VT). HTRF ratio (665/620) was converted to cAMP concentration using a cAMP standard.

Matrix metallopeptidase (MMP) zymography

Freshly differentiated BMDMs were cultured in bone marrow macrophage-differentiation media until 80% confluence. Cells were starved for 24h in IMDM medium and incubated with PGE2 (500 nM) plus vehicle, OA (2.5 µM) or NO2-OA (2.5 µM) for an additional 12h. Then, the supernatant was collected and centrifuged to remove any residual debris. MMP2 and MMP9 activity was assessed by Zymography of the supernatant in 1% gelatin gel, followed by staining with Coomassie blue, as previously described [41].

In silico ligand-receptor docking simulation

A modified crystal structure of the human EP4 receptor at 3.2 Å resolution has been determined [42]. Protein-ligand docking simulation of NO2-OA and EP4 was performed using an EADock DSS based online interface, SwissDock [43, 44]. Binding energies were determined using a CHARMM-based function [45].

Statistical analysis

Statistical analysis was performed using GraphPad Prism 8.3.0 or R-3.6.2. Data were tested for normality using the Shapiro-Wilk test and tested for “normality vs. lognormality” when normalized over control. For data that passed the normality test, the residual plot was used to ensure the homogeneity of variance. Weighting was performed when necessary. ROUT with Q set at 1% was used to identify outliers for raw data. Unpaired Student t-test was used to compare the difference between two means, one-way ANOVA was used to compare means from more than two groups, and nonlinear regression or two-way ANOVA was used for data with two independent variables. Tukey’s or Sidak post hoc test was added to compare individual means. For data that failed the normality test, the Mann-Whitney U test or Kruskal-Wallis test followed by Dunn’s comparisons were used as an alternative. Mantel-Cox test was used for the survival assay. For non-linear regression, three or four parameters, global or separate fitting, and constraints were compared in Prism. For incidence analysis, 2X3 Fisher’s exact test following the post hoc test was conducted in R using the “rcompanion” package. Tests were performed in two-tail except for supplementary figure 6A, in which treatment higher than control was chosen. Unless otherwise stated, continuous variables were all presented as mean ± standard error of the mean (SEM). Results with p<0.05 for both the main test and the post hoc test were considered as statistically significant. All in vitro results were representative of at least three independent experiments.

Power analysis

Power analysis was used to estimate the number of mice needed for the study based on the effect size and S/N ratio of a pilot study to achieve a power of 0.8 at α=0.05 (https://www.stat.ubc.ca/~rollin/stats/ssize/n2.html )

Justification of sex factors

Samples from males and females were used in our studies to minimize the impact of sex differences. However, in the AngII- and hypercholesterolemia-induced AAA mouse model, we were limited to male mice because, in prior experiments, less than 10 percent of female mice developed AAA with this model (data not shown).

Results

Nitro-oleic acid attenuates AAA formation in the mouse model

We first tested whether NO2-OA could protect against AAA development in an Ang II plus hypercholesterolemia model of AAA in C57BL/6 wild type mice. Three groups of mice (n=25 to 30 per group) received a chronic subcutaneous infusion of either vehicle control (PEG-400), OA, or NO2-OA, (Figure 1A). After 4-weeks, a significant decrease in AAA incidence was observed in NO2-OA treated mice when compared to vehicle (28.6% vs. 68.4%, p=0.0117) or OA (28.6% vs. 69.6%, p=0.0078) treated group (Figure 1B, C). This effect was accompanied by a significant decrease in the maximal diameter of the suprarenal aorta region in the NO2-OA treated group when compared to the vehicle (1.16±0.09 mm vs. 1.65±0.17 mm, p=0.0289) or OA (1.16±0.09 mm vs. 1.78±0.18 mm, p=0.0121) treatment groups (Figure 1D). While ECM degradation is prominent in the vehicle control and OA-treated mice, NO2-OA-treatment was protective, maintaining vascular integrity and significantly reduced elastin fiber degradation (p=0.0274 vs. vehicle and p=0.0304 vs. OA, Figure 1E, F). Metabolic changes did not account for the NO2-OA protective effect as no changes in cholesterol or body weight were observed [F(2,67)=1.225, p=0.3003 for total cholesterol level and F(2,55)=2.866, p=0.0655 for body weight, Supplementary Table S3]. While NO2-OA significantly decreased the serum TG levels when compared to vehicle (p=0.0048), this decrease was not specific as TG levels in OA-treated mice were significantly lower than the vehicle (p=0.0136), and no significant difference in serum TG levels was observed between the NO2-OA and OA groups (p=0.9458, Supplementary Table S3). Overall, these results show specific protection by NO2-OA against AAA development in vivo, an effect that is not shared by OA.

Fig. 1. Characterization of the protective effect of NO2-OA in the Ang II plus hypercholesterolemia-induced AAA mouse model.

Fig. 1.

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(A) Schematic illustration of the in vivo experimental design. Ten week old male C57BL/6 mice were IP injected with AAV carrying PCSK9 gain-of-function (D377Y) mutation and fed a western diet. After two weeks, each mouse was implanted with a pump delivering AngII at a rate of 1,500 ng/kg/min plus an additional pump containing either PEG-400 (vehicle), OA or NO2-OA with a delivery rate of 5 mg/kg/day (n=25 to 30 per group) and carried to the endpoint for another four weeks. Mice with ruptured aorta within the first week or with total plasma cholesterol less than 250 mg/dl were excluded from the study. (B) Representative morphological differences of the abdominal aorta. (C) AAA incidence calculated by using 1.2mm as the cutoff diameter indicated by the dotted line in (D). (D) Average maximal diameter of the suprarenal aorta region. (E) Representative H&E and VVG staining of the paraffin-embedded suprarenal aortic cross-sections (5µm). (F) Grade of elastic fiber degradation in the aorta (1 to 4). A 2X3 Fisher’s exact test followed by posthoc test was performed for (C). For (D) and (F), the Kruskal-Wallis test followed by Dunn’s multiple comparisons was performed. Continuous data are presented as mean ± SEM. Scale bar = 20 µm for (E). A p< 0.05 for both the main test and post hoc test was considered statistically significant.

Nitro-oleic acid prevents leukocyte/macrophage infiltration in the vasculature

The contribution of infiltrated leukocytes/macrophages in the vasculature to AAA development has long been demonstrated [6, 9]. Hence, we tested the effect of NO2-OA treatment on the infiltration process in vivo by evaluating the expression of several genes related to monocyte/endothelial cell-cell interaction. Mice treated with NO2-OA had significantly decreased expression of the vascular cell adhesion protein 1 (Vcam1, p=0.0008) and Monocyte chemoattractant protein-1 (MCP1, p=0.0003) when compared with the vehicle control group in the suprarenal aortic tissue (Figure 2A). The expression of interleukin 6 (Il6, p=0.1852) and intercellular adhesion molecule 1 (Icam1, p=0.2612) in the NO2-OA group were reduced but did not reach statistically significance compared to vehicle. These local responses were also observed systemically, where protein levels of both circulating MCP1 (p=0.0006) and IL6 (p=0.0459) were significantly decreased by NO2-OA (Figure 2B, C). In order to evaluate the role of infiltrating leukocytes and macrophages in the inflammatory responses, the suprarenal aorta was digested into single cells, and the number of leukocytes (CD45+), total macrophages(CD64+) and M1 like macrophages (CD64+, CD11c+) were determined (Supplementary Figure1A, Figure 2D). Compared with the vehicle group, significantly fewer leucocytes (CD45+) appeared in the vasculature upon NO2-OA treatment (14.87±3.28% vs. 50.03±13.02% in vehicle control, p=0.0489, Figure 2E). Notably, the abundance of pro-inflammatory M1 like macrophages (CD64+, CD11c+) was also decreased in the NO2-OA group (3.27±0.42% vs. 11.43±2.65% in vehicle control, p=0.0338, Figure 2F), which is consistent with the previous observation that Nitro-FAs affect the differentiation, adhesion and polarization of macrophages [31–33]. Additionally, we treated HUVECs with Ang II for 6h and used the conditioned medium to induce migration of RAW 264.7 cells (Supplementary Figure 2A). We observed that NO2-OA significantly inhibits endothelial-dependent macrophage migration in the presence of Ang II but not under basal conditions (vehicle, p=0.0446, Supplementary Figure 2B). These data indicate that NO2-OA is effective in limiting endothelial-dependent leukocyte/macrophage migration to the AAA lesion area.

Fig. 2. NO2-OA treatment suppressed leukocytes/macrophages infiltration in the vasculature.

Fig. 2.

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(A) Quantification of gene expression levels in the mice suprarenal aorta region by real-time PCR (n=3 to 4). (B, C) Serum cytokines levels measured by ELISA (n=8 to 11). (D) FACS of single cells isolated from the mice suprarenal aorta region. Each sample is the pool of cells from three mice (n=3). (E, F) Quantification of the percentage of CD45+ leukocytes and CD64+, CD11c+ M1-like macrophages in total cells from the FACS analysis. Ordinary one-way ANOVA followed by Tukey’s test was performed for (A), (E) and (F). For ELISA in (B) and (C), to avoid the effect of the non-detected data point, a non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons was performed by adding zero to the missing data point. Data presented as mean ± SEM. A p< 0.05 for both the main test and post hoc test was considered statistically significant.

Nitro-oleic acid inhibits ox-LDL induced NF-κB activation and pro-inflammatory responses in part via activation of PPARγ

While the role of oxLDL in the initiation and development of atherosclerosis is well established, a recent study utilizing a systematic approach suggested that oxLDL also participates in the formation and development of AAA via activation of inflammatory responses [46]. Thus, we hypothesized that NO2-OA exerts a protective role through the inhibition of the oxLDL-induced macrophage inflammatory activation. Here, BMDMs were treated with oxLDL (50μg/ml), and oxLDL-induced p65 nuclear translocation was evaluated in nuclear and cytoplasmic fractions (Figure 3A, B). We found that NO2-OA significantly inhibited oxLDL-induced p65 nuclear translocation, with a nuclear to cytoplasmic ratio of 14.10±1.92 vs. 27.93±1.89 in the control group (p=0.0238, Figure 3C). Further analysis of inflammatory cytokines in the medium from oxLDL-treated cells showed a significant decrease in IL6 level (p=0.0327 vs. control) and a marginally significant reduction in MCP1 level (p=0.0664 vs. control). However, no significant difference was observed in TNFα levels, as compared to the control group (p=0.3290, Figure 3D–F. PPARγ expression in monocytes/macrophages is upregulated by oxLDL, which may amplify its role under disease conditions like those predisposing for atherogenesis and AAA development [47]. We showed that oxLDL-induced expression of the pro-inflammatory genes Il1b, and Mmp9, which are responsible for ECM degradation, were significantly decreased by NO2-OA treatment (p<0.0001 vs. control), and that the protective effect of NO2-OA was partially blocked by giving an irreversible PPARγ antagonist, GW9662 (p=0.0310 for Il1b, and p=0.0321 for Mmp9, Figure 3G), or siPPARγ (Supplementary Figure 3A), indicating that PPARγ signaling is partially responsible for the protective effect of NO2-OA on ox-LDL-induced pro-inflammatory responses.

Fig. 3. NO2-OA prevents oxLDL-induced NF-κB activation and pro-inflammatory cytokine production.

Fig. 3.

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Primary BMDMs were pretreated with vehicle, OA (2.5 µM) or NO2-OA (2.5 µM) for 1h, followed by treatment of oxLDL (50 to 100 µg/ml) for the indicated times. (A, B) Nuclear (A) and cytoplasmic protein (B) fractions were isolated 1h after oxLDL treatment and subjected to western blot with antibodies against p65, Lamin a/c (nuclear marker) and GAPDH (cytosolic marker). Representative western blot images of three independent experiments are shown. (C) Quantification of the p65 nuclear to cytoplasmic ratio by image studio (normalized by the internal control, n=3). (D–F) Following 8h of oxLDL treatment, levels of IL6, MCP1 and TNFα in the cell medium were measured by ELISA (n=4). (G) Real-time PCR quantification of gene expression levels of IL1b and Mmp9 in total mRNA extracted from the BMDMs. (C) Ordinary one-way ANOVA followed by Tukey’s test was performed. As of (D–F), the basal level contains several non-detectable values, so ordinary one-way ANOVA followed by correction of FDR were performed for the oxLDL-treated groups only. Ordinary one-way ANOVA followed by Tukey’s test was performed for (G). Data presented as mean ± SEM. A p< 0.05 for both the main test and post hoc test was considered statistically significant.

Nitro-oleic acid is a biased regulator of PGE2-dependent EP4 signaling

To address other pathways potentially contributing to the NO2OA protective effects, we investigated the PGE2-dependent EP4 signaling. It has been reported that a signaling axis involving the macrophage cyclooxygenase 2 (COX2), the microsomal isoform of prostaglandin E synthase 1 (mPGES1) and EP4 contributes to AAA development (Supplementary Figure 4A) [48]. For example, COX2, mPGES-1, and EP4 levels are upregulated at human aneurysm lesion sites [48, 49]. Similar upregulation was also seen in human thoracic aneurysm lesions, and the suprarenal aorta region of the AngII plus hypercholesterolemia-induced murine AAA model (Supplementary Figure 4B, C). EP4 receptors are highly expressed in macrophages and are responsible for PGE2-dependent upregulation of MMP secretion [49, 50]. By overexpressing EP4 in 293T cells, we demonstrated that NO2-OA significantly rightward shifted the dose-response curve over a range of concentrations of the EP4 agonist PGE2, with a best-fit EC50 of 1.2 nM vs.109.5 pM in the control group (p=0.0069), as did L-902,688, a highly specific EP4 agonist, with a significant rightward shift (best-fit EC50 of 724.3 pM vs. 932.2 pM in the control group, p=0.0483, Figure 4A).

Fig. 4. NO2-OA inhibits PGE2-dependent EP4 cAMP downstream signaling.

Fig. 4.

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(A) Three parameters nonlinear regression of EP4 global fitting dose-response curve. The SEAP-EP4 reporter was overexpressed in 293T cells and the NO2-OA induced EC50 shift was calculated using PGE2 as an agonist. (B) HTRF assay of cAMP-Gs coupled receptor. Inhibition of PGE2-induced cAMP recruitment (at IC90 10 nM) by various doses of NO2-OA in 293T cells overexpressing EP4. (C) Modeling of the predicted binding site of NO2-OA and PGE2 to the EP4 receptor with the highest rank score. (D) Representative figure of gelatin zymography indicating MMP 2/9 activity. BMDMs were incubated with L-902,688 (100 nM) plus vehicle, OA (2.5 µM) or NO2-OA (2.5 µM) for 12h, and MMP 2&9 activity was measured by gelatin zymography. For (A) and (B), three or four parameters, global or separate fitting, constraints, and the significance of EC50 were determined in Prism. Data presented as mean ± SEM. A p < 0.05 was considered statistically significant.

Moreover, NO2-OA dose-dependently reduced PGE2 -induced (IC90 10nM) recruitment of cAMP to the EP4 Gs-coupled receptor with a best-fit IC50 at 2.8 μM (R2=0.9142, Figure 4B). Molecular-receptor docking results showed that the predicted binding site of NO2-OA with the highest score is close to the orthosteric binding pocket for PGE2 (Figure 4C). MMPs, especially MMP2/9, play essential roles in ECM degradation and AAA development [6]. We then used gelatin zymography to determine the activity of macrophage MMP2/9 upon NO2-OA treatment. In BMDMs, PGE2 upregulates MMP9 activity, and this effect can be significantly diminished by NO2-OA treatment, while no significant changes were observed in MMP2 activity (Supplementary Figure 5). The decreased MMP9 by NO2-OA was further confirmed by using 100 nM L-902,688, suggesting an EP4 specific effect (Figure 4D). Aside from its positive contribution to ECM degradation, the PGE2-dependent activation of the EP4 receptor exerts anti-inflammatory actions by recruiting EPRAP, which inhibits NF-κB and MEK activation through binding to p105 and preventing its phosphorylation [51]. Herein, our results demonstrated that NO2-OA does not suppress the PGE2-mediated protective effect against LPS-induced macrophage activation, as reflected by the expression of Tnf, Il6, and Ccl4 (Supplementary Figure 6A). Moreover, Co-IP assay in cells overexpressing HA-tagged EPRAP (FEM1A), and p105 showed that up to 5 μM NO2-OA treatment had no significant effect on the ERRAP/P105 interaction (Supplementary Figure 6B, C and 7). Overall, these results indicate that NO2-OA serves as a biased regulator of EP4-dependent PGE2 signaling.

Discussion

In spite of the urgent unmet need for effective pharmaceutical management of AAA, recent clinical trials of AAA therapies using small molecule drug candidates targeting individual factors like blood pressure, plasma cholesterol level, vascular inflammation and ECM degradation failed to provide a satisfying outcome [9]. At the same time, multiple studies and clinical trials have demonstrated the participation and active contribution of monocytes/macrophages to all stages of AAA development, involving distinct mechanisms that mediate the recruitment of immune cells, activation of pro/anti-inflammatory responses and degradation of the aortic wall [8]. Nitro-FAs, endogenous mediators of diverse inflammatory and metabolic cell signaling and gene expression responses, have displayed various beneficial effects in vascular disease and monocytes/macrophages activation [31–33].

CXA-10, a synthetic homolog of one specific regioisomer of NO2-OA, is presently being studied in phase II clinical trials for treatment of a chronic inflammatory-related renal disorder (focal segmental glomerulosclerosis) and pulmonary arterial hypertension (NCT04053543, NCT03422510). Herein, we investigated the effect of NO2-OA in an AngII/PCSK9-induced AAA mouse model and found that NO2-OA could attenuate AAA formation. The pathological assessment uncovered a significantly suppressed suprarenal aneurysm enlargement and less elastin degradation (Figure 1), highlighting the protective role of NO2-OA and other electrophilic FAs in AAA development.

Vascular infiltration by leukocytes and macrophages, an essential feature and contributor to AAA development, was significantly downregulated by NO2-OA. This effect is possibly due to the simultaneous downregulation of adhesion molecules expression and chemoattractant secretion (Figure 2). Furthermore, in vitro studies using biologically relevant stimuli demonstrated a protective role of NO2-OA, including reduced migration of macrophages upon AngII-induced endothelial stimulation. Moreover, potent inhibition of oxLDL-induced NF-κB activation and nuclear translocation of the p65 transcription factor with NO2-OA treatment was observed. This effect was mediated, at least partially, by the activation of PPARγ and inhibited by the irreversible PPARγ inhibitor GW9662 (Figure 3).

Representing a convergence of lipophilic and electrophilic chemical structures, NO2-FAs not only function on intracellular nuclear receptors but also modulate membrane-associated proteins such as Toll-like receptors [32]. Herein, we also revealed the inhibition of PGE2-induced EP4 activation by NO2-OA (Figure 4). It is widely accepted that the macrophage COX2-PGE2-EP4 axis participates in the etiology of human AAA disease [48]. Studies using different EP4-specific antagonists have demonstrated protection against AAA formation in the AngII/Apoe−/− and CaCl2-induced AAA models in mice, with decreased MMP production and activity as common features [49, 52, 53]. It was previously reported that a cAMP-PKA/PI3K-Akt cascade is responsible for the MMP9 produced in response to EP4 activation. Forskolin, an activator of adenylate cyclase (AC) upregulates MMP9 expression, while SQ22536, an inhibitor of AC, inhibited the MMP9 expression. Results from both activation of cAMP and inhibition of cAMP provided substantial evidence that cAMP regulates MMP9, and thus it is reasonable to assume that inhibition of EP4 by NO2-OA operates through decrease of cAMP [54]. However, EP4 deficiency in bone marrow-derived cells was found to increase inflammation and AAA incidence [55]. These conflicting observations could be a consequence of differential effects on the downstream signaling pathways since the Gs-coupled EP4 not only facilitates cAMP recruitment but also counteracts NF-κB activation through the formation of the EPRAP/p105 complex [51]. In this regard, we demonstrated that NO2-OA functions as a biased regulator of EP4 signaling responses by increasing the EP4-dependent anti-inflammatory effects of PGE2 induced by LPS treatment without interfering with the EPRAP/p105 interaction (Figure S5).

The chemical nature of NO2-OA renders it capable of post-translational modification of cysteine residues found in numerous regulatory proteins including AT1 receptor, thus brining the concerns that altered blood pressure may also contribute to the protective effect of NO2-OA [25]. Although this is true for low AngII (500 ng/kg/day) condition, in our AAA model, we used a relatively high dose of AngII (1500 ng/kg/day) and observed no significant decrease in blood pressure under this specific experimental setting at the doses of NO2-OA required to see a reduction in AAA (Supplementary Table S3). Meanwhile, in healthy human volunteers (NCT02313064, NCT02127190), administration NO2-OA also showed no reduction in blood pressure. These data indicate that the observed protective effects of NO2-OA on AAA are unlikely due to changes in blood pressure.

In conclusion, we reveal a protective effect of the electrophilic NO2-OA against macrophage activation and AAA development (Figure 5). Our results underscore a potential new clinical therapeutic opportunity for NO2-FAs, such as the nitroalkene derivative of oleic acid used herein, in the treatment of AAA. In addition, our findings provide a rationale for using multi-targeted, pleiotropic drugs to treat this pathologically-diverse and complex disease. Future studies will be needed to establish whether NO2-FAs are able to ameliorate established AAA, by attenuating or even reversing the progression of this devastating disease.

Fig. 5.

Fig. 5.

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Graphic summary of the study.

Supplementary Material

Supplementary Material

Acknowledgments and Authors’ contribution:

Y. Zhao, Z. Chang, G. Zhao, and J. Zhang performed experiments and analyzed results; Y. Zhao, J. Zhang, and Y.E. Chen wrote the paper; H. Lu, W. Xiong, W. Liang, H. Wang, L. Villacorta, T. Zhu, Y. Guo, Y. Fan, L. Chang, M.T. Garcia-Barrio, F.J. Schopfer, B.A. Freeman, J. Zhang provided technical support and discussed the project; J. Zhang and M.T. Garcia-Barrio did critical editing of the manuscript; J. Zhang and Y.E. Chen led the experimental design.

Funding: This work was supported by grants to E. Chen (R01-HL068878), J. Zhang (R01-HL138139), L. Villacorta (R01-HL123333), Y. Zhao (Rackham Graduate Student Research Grants), B.A. Freeman (P01-HL103455), and the Mouse Metabolic Phenotyping Center at Michigan (MMPC, NIH U2CDK110768).

Footnotes

Conflicts of interest/Competing interests: FJS, BAF, and YEC acknowledge an interest in Complexa, Inc and FJS and BAF in Creegh Pharmaceuticals, Inc.

Ethics approval: The collection of human samples was approved by the Institutional Review Board (Hum00077616).

Consent to participate: Informed consent was obtained from all individual participants included in the study.

Consent for publication: Consent for publication was acquired from all individual participants.

Availability of data and material: Additional data and material information are available upon request.

Code availability: All codes are available upon request.

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