Novel gene regulatory networks identified in response to nitro-conjugated linoleic acid in human endothelial cells

Abstract

Endothelial cell (EC) dysfunction is a crucial initiation event in the development of atherosclerosis and is associated with diabetes mellitus, hypertension, and heart failure. Both digestive and oxidative inflammatory conditions lead to the endogenous formation of nitrated derivatives of unsaturated fatty acids (FAs) upon generation of the proximal nitrating species nitrogen dioxide (·NO₂) by nitric oxide (·NO) and nitrite-dependent reactions. Nitro-FAs (NO₂-FAs) such as nitro-oleic acid (NO₂-OA) and nitro-linoleic acid (NO₂-LA) potently inhibit inflammation and oxidative stress, regulate cellular functions, and maintain cardiovascular homeostasis. Recently, conjugated linoleic acid (CLA) was identified as the preferential FA substrate of nitration in vivo. However, the functions of nitro-CLA (NO₂-CLA) in ECs remain to be explored. In the present study, a distinct transcriptome regulated by NO₂-CLA was revealed in primary human coronary artery endothelial cells (HCAECs) through RNA sequencing. Differential gene expression and pathway enrichment analysis identified numerous regulatory networks including those related to the modulation of inflammation, oxidative stress, cell cycle, and hypoxic responses by NO₂-CLA, suggesting a diverse impact of NO₂-CLA and other electrophilic nitrated FAs on cellular processes. These findings extend the understanding of the protective actions of NO₂-CLA in cardiovascular diseases and provide new insight into the underlying mechanisms that mediate the pleiotropic cellular responses to NO₂-CLA.

INTRODUCTION

The vascular endothelium maintains vessel structure and adapts to hemodynamic alterations and chemical signals by the production of diverse factors involved in vascular homeostasis, cellular adhesion, thromboresistance, and vessel wall inflammation. Numerous risk factors such as smoking, hypercholesterolemia, and diabetes mellitus lead to endothelial cell (EC) dysfunction (56, 63), which is an early mediator in the development of atherosclerosis.

Nitro-fatty acids (NO₂-FAs) contribute to the maintenance of cell homeostasis. Inflammatory conditions lead to nitric oxide (NO) and nitrite ($N{O}_2^{-}$)-dependent unsaturated FA nitration, resulting in the endogenous formation of both free- and esterified-FA nitroalkene derivatives (4, 12, 17, 59) of linoleic acid (NO₂-LA) and oleic acid (NO₂-OA). NO₂-FAs protect against cardiac ischemic injury (47), angioplasty-induced restenosis (8), cardiac ischemia-reperfusion (I/R) injury (39), angiotensin II-induced hypertension (69), and atherosclerosis (46). The specific regioisomer 10-nitro-OA (CXA-10) is currently being evaluated in Phase II clinical trials in pulmonary arterial hypertension (NCT03449524) and primary focal segmental glomerulosclerosis (NCT03422510). NO₂-FA adduction to nucleophilic amino acids (His and Cys) in redox-sensitive enzymes and signaling mediators alters protein functions, including inactivation of the oxidant-generating enzyme xanthine oxidoreductase (XOR) (27), activation of nuclear factor 2-related factor (Nrf2)-dependent phase 2 gene expression via S-nitroalkylation of Keap-1 (24) and S-nitro-alkylation of the NF-κB p65 subunit (10). Posttranslational protein modifications induced by NO₂-FAs alter the target protein structure and functions, thereby regulating critical biological processes in cells (28, 58).

Recently, conjugated linoleic acid (CLA) was identified as a preferential FA substrate for nitration in vivo (4, 12, 49). Endogenously occurring nitro-CLA (NO₂-CLA) exists as two predominant positional isomers: 9- and 12-NO₂-CLA (4, 55). Oral consumption of CLA in combination with dietary sources of $\text{N}{\text{O}}_2^{-}$ leads to NO₂-CLA formation that reaches clinically relevant plasma levels in mice (4) and humans (12, 20). The abundance of CLA in vivo and the preferential nitrogen dioxide reaction with conjugated double bond configurations result in NO₂-CLA being readily produced endogenously and an attractive target for potential clinical applications (12, 20, 55). However, the biological signaling actions of NO₂-CLA in ECs remain to be investigated.

In the present study, we revealed a distinct transcriptome regulated by NO₂-CLA in primary human coronary artery endothelial cells (HCAECs) with RNA sequencing (RNA-Seq). Analysis of differential gene expression and the engagement of critical signaling pathways reveals that NO₂-CLA mediates diverse effects on multiple endothelial cell functions and regulatory networks.

MATERIALS AND METHODS

Cell culture.

HCAECs isolated from a male, 37 yr old, nonsmoking donor were purchased from Lonza (#CC-2585) and cultured in the EGM-2MV microvascular endothelial cell growth medium-2 containing 5% fetal bovine serum (Lonza, #CC-3202) at a 37°C/5% CO₂-humidified incubator. HCAECs for the treatments and analysis in this study were used at passages 3–5.

Cell treatment.

For the RNA-Seq experiment, two sets of HCAECs at 90% confluence were treated with DMSO, CLA (10 µM), or NO₂-CLA (10 µM) for 1 h, followed by addition to one of the sets of either ethanol (Control) or palmitic acid (200 µM) (Cayman Chemical) for the subsequent 6 h. For RNA-Seq analysis, six groups were considered: DMSO + control (ethanol), CLA (10 µM) + control, NO₂-CLA (10 µM) + control, DMSO + palmitic acid, CLA (10 µM) + palmitic acid, and NO₂-CLA (10 µM) + palmitic acid. Each group had four biological replicates. Next, quantitative real-time PCR (qPCR) was applied to validate the genes identified from RNA-Seq and establish the dose-dependent gene regulation in response to NO₂-CLA treatment as follows: HCAECs at 90% confluence were treated with DMSO, different concentrations of CLA (1, 5, 10 µM), or NO₂-CLA (1, 5, 10 µM) for 1 h, followed by addition of either ethanol or palmitic acid (200 µM) for subsequent 6 h. NO₂-CLA was synthesized as described previously (65).

RNA extraction.

Total RNA from HCAECs was extracted with RNeasy Mini Kit (Qiagen) and treated on-column with RNase-free DNase I following the manufacturer’s protocol (Qiagen). Total RNA samples were assessed for quality with the BioAnalyzer (Agilent).

RNA-Seq.

RNA library preparation and sequencing were performed by the DNA sequencing core of the University of Michigan. Briefly, the RNA library was prepared with TruSeq RNA Library Prep Kit v2 (Illumina, WI) and sequenced on a HiSeq 4000 platform (Illumina) to generate nonstranded single end 51 bp reads according to the manufacturer’s protocols (35). In total, ~1,030 million reads were generated with the HiSeq 4000 platform (average 43 million reads per sample). The raw data from this study have been submitted to the National Center for Biotechnology Information Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE124522.

Read mapping and gene expression analysis.

The quality control of the sequencing reads from each sample was assessed with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). One sample from the control group was excluded from the subsequent analysis because the quality control analysis of this sample showed low mapping rate due to low library quality. The human reference cDNA sequences were downloaded from the Ensembl database (GRCh38 ftp://ftp.ensembl.org/pub/release-93/fasta/homo_sapiens/cdna/). The reference was indexed with Salmon, and gene expression quantification was performed with Salmon in its nonalignment-based mode (42). Differential expression analysis was performed with the DEseq2 package in R (37). The log2 fold change was calculated by default settings with the DESeq2 package, in which the log2 fold change was estimated using the empirical Bayes shrinkage strategy that shrinks log2 fold changes (LFC) estimates toward zero when the gene expression is low (37). Differentially expressed genes (DEGs) are defined as >1.3-fold change in expression level and false discovery rate <0.05. principal component analysis (PCA), volcano plot, and heat maps were generated with an in-house R script.

Gene ontology and KEGG pathway enrichment analysis.

The Gene Ontology and KEGG pathway enrichment analyses were performed with the ClueGO package in Cytoscape (3). Multiple testing correction was performed using Benjamini and Hochberg’s approach (1).

qPCR.

Total RNAs were reverse-transcribed into cDNA with random hexamers used as primers (SuperScript III RT-PCR kit, ThermoFisher). qPCR was performed with iQ SYBR Green Supermix (Bio-Rad) as a validation set. Gene expression was normalized against the internal control glyceraldehyde 3-phosphate dehydrogenase. The primer sequences are shown in Supplemental Table S1. The supplemental tables are available at https://zenodo.org/record/2530146#.XOKvu0hKiUl.

Statistical analysis.

Statistical analyses for qPCR were performed by one-way ANOVA followed by Dunnett test for multiple comparisons with GraphPad Prism version 6.0. Quantitative data are expressed as means ± SE. A P value of < 0.05 was considered statistically significant.

RESULTS

DEGs in response to NO₂-CLA in HCAECs.

To investigate the effect of NO₂-CLA on the EC transcriptome, we treated the HCAECs with electrophilic NO₂-CLA (10 µM), nonelectrophilic CLA (10 µM), or DMSO. Differential gene expression was pairwise compared between CLA versus DMSO, NO₂-CLA versus DMSO, and NO₂-CLA versus CLA (Fig. 1A). Notably, only 150 genes show significant changes between CLA versus DMSO (Supplemental Table S2). In sharp contrast, a total of 1,143 DEGs were identified in response to NO₂-CLA treatment (Fig. 1B) with 298 genes overlapping between the comparisons of NO₂-CLA versus DMSO and NO₂-CLA versus CLA (Supplemental Tables S3 and S4). Pearson’s correlation coefficient matrix (Fig. 2A), PCA (Fig. 2B), and heat map (Fig. 2C) based on the DEGs of each group consistently show that NO₂-CLA induces a pattern of gene expression profile distinct from DMSO and CLA treatment.

Fig. 1.

Genes regulated by nitro-conjugated linoleic acid (NO₂-CLA) in human coronary artery endothelial cells (HCAECs). A: pairwise volcano plot of differentially expressed genes (DEGs) between different treatments. B: Venn diagram showing overlapping genes between NO₂-CLA versus DMSO and NO₂-CLA versus CLA. DEGs are defined as >1.3-fold change (upregulated, red; downregulated, green) and false discovery rate < 0.05, n = 4 per group.

Fig. 2.

Differentially expressed genes (DEGs) in HCAECs. A: correlation matrix (Pearson correlation coefficients) of all samples. B: principal component analysis of the DEGs. C: heat map and hierarchical clustering of the DEGs between different treatments. Color bar [red to blue (3 to −3) vertical bar] denotes the row-scaled transcripts per kilobase million (TPM) value, representing the Z score of gene expression across samples.

qPCR validation of RNA-Seq results.

To validate the results from RNA-Seq in HCAECs, we chose the top genes (6 upregulated and 6 downregulated) with the lowest P value from the overlapping DEGs between NO₂-CLA versus DMSO and NO₂-CLA versus CLA (Fig. 3A) and used qPCR to determine their expression in the different groups in the different groups and in response to increasing doses of NO₂-CLA (Fig. 3B). The qPCR results validated the gene expression changes. Moreover, these genes responding to NO₂-CLA were expressed in a dose-dependent manner in ECs (Fig. 3). In the DEGs, heme oxygenase 1 (HMOX1), oxidative stress-induced growth inhibitor 1 (OSGIN1), and matrix metallopeptidase 1 (MMP1) were increased by 91 ± 6.4-, 12.5 ± 2.1-, and 3.2 ± 0.23-fold, respectively, after NO₂-CLA treatment (10 µM). Endothelin-1 (EDN1) was decreased by 31 ± 2.5% in NO₂-CLA (10 µM)-treated ECs.

Fig. 3.

Validation of the RNA sequencing (RNA-Seq) by quantitative real-time PCR (qPCR). A: expression of the six most significantly upregulated and downregulated genes as determined by RNA-Seq. Data are shown as fold change in TPM. B: HCAECs were treated with DMSO, CLA, and NO₂-CLA at different doses (1, 5, or 10 μM). Fold change in mRNA levels of the indicated genes was determined by qPCR relative to the DMSO control set as 1. qPCR data shown are representative of 3 independent experiments and normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are presented as means ± SE, n = 4 per group. *P < 0.05; **P < 0.01 by one-way ANOVA followed by Dunnett test, compared with the DMSO group.

Pathway enrichment analysis of DEGs.

To define the biological actions of NO₂-CLA in ECs and identify novel underlying molecular mechanisms, we pursued the pathway enrichment analysis of DEGs. Based on the overlapping DEGs from the two comparisons NO₂-CLA versus DMSO and NO₂-CLA versus CLA, we identified the overrepresented pathways and novel NO₂-CLA-regulated genes. The top enriched pathways in response to NO₂-CLA were related to cell cycle regulation, ATPase activity, heat shock protein, and nuclear chromosome (Fig. 4, A and B; Supplemental Table S5). Furthermore, we revealed two overrepresented pathways including fluid shear stress and atherosclerosis (P = 0.00148) and response to hypoxia (P = 0.0472) that are known to be crucial for endothelial function (Fig. 4C). Among the components of these pathways, we identified novel NO₂-CLA-responsive genes and validated them by qPCR (Fig. 4D). NO₂-CLA increased the expression of glypican 1 (GPC1) and Krüppel-like factor 2 (KLF2) by an average of 1.98- or 1.31-fold, respectively. Also, NO₂-CLA decreased bone morphogenic protein-4 (BMP4) and caspase-1 expression by an average of 38 or 26%, respectively.

Fig. 4.

Function and pathway enrichment of DEGs in the groups treated with DMSO, CLA (10 μM), or NO₂-CLA (10 μM). A: top GO terms and KEGG pathways in the overlapping DEGs identified from the comparisons of NO₂-CLA, DMSO, and CLA. B: network of the top GO terms and pathways. C: heat map of the DEGs in two pathways essential to endothelial cell (EC) function. D: qPCR validation of the novel NO₂-CLA responsive genes in the pathways in C. data are presented as means ± SE, n = 4 per group. *P < 0.05; **P < 0.01 by one-way ANOVA followed by Dunnett test, compared with the DMSO group.

NO₂-CLA prevents palmitic acid-induced inflammatory responses in HCAECs.

The high proportion of saturated FA consumption is associated with insulin resistance, liver steatosis, and cardiovascular disease (CVD) (5, 52). As a saturated FA, palmitic acid can induce inflammatory responses in vascular ECs (23, 33). To determine whether NO₂-CLA antagonizes adverse palmitic acid-induced effects on HCAECs, we compared the response to NO₂-CLA and DMSO in cells subjected to palmitic acid (200 μM). We identified a distinct transcriptomic pattern of 87 genes that were influenced by palmitic acid treatment and in turn modulated by concurrent NO₂-CLA treatment (Fig. 5A, Supplemental Table S6). Pathway enrichment analysis revealed that some of these genes belong to proinflammatory pathways such as TNF signaling pathway, leukocyte adhesion to vascular endothelial cell, NF-κB signaling pathway, and cellular extravasation. These proinflammatory pathways are among the top pathways with the lowest P value (Fig. 5, B and C). For example, in the NF-κB pathway (P = 0.00296), NO₂-CLA inhibited palmitic acid-induced proinflammatory adhesion molecules such as VCAM-1, SELE, and ICAM-1 expression (Fig. 5, D and E).

Fig. 5.

NO₂-CLA prevents palmitic acid (PA)-induced endothelial inflammation. HCAECs were pretreated with DMSO or NO₂-CLA (10 μM) for 1 h and then treated with PA (200 μM) or ethanol control for another 6 h. A: heat map of PA-regulated genes protected by NO₂-CLA. B: top GO terms and KEGG pathways in the NO₂-CLA reversed genes. C: group of the top GO terms. D: heat map of the effects of NO₂-CLA on the NF-κB pathway response to palmitic acid in ECs. E: fold change in mRNA levels of the selected proinflammatory adhesion molecules in the indicated experimental conditions. mRNA levels were determined by qPCR and normalized by GAPDH with fold change expressed relative to the DMSO group, set as 1. The mRNA abundance of these proinflammatory adhesion molecules was determined by qPCR. Data are presented as means ± SE, n = 4 per group. *P < 0.05; **P < 0.01 by one-way ANOVA followed by Dunnett test, compared with the DMSO group.

DISCUSSION

Electrophilic NO₂-FAs display signaling responses following Michael addition with kinetically susceptible nucleophilic amino acids in target proteins, resulting in a broad array of responses in diverse cell types and tissues (58). NO₂-OA and NO₂-LA mediate adaptive anti-inflammatory and metabolic effects in ECs, macrophages, and vascular smooth muscle cells (51, 58). NO₂-CLA constitutes a new class of NO₂-FAs in addition to NO₂-OA and NO₂-LA. To better define potential mechanisms accounting for endogenously generated NO₂-CLA actions in CVDs, we performed RNA-Seq analysis in primary human ECs. Our data indicate that NO₂-CLA induces profound changes in the EC transcriptome and thus provides insight into the pathways and networks responding to this and other electrophilic fatty acid nitroalkene derivatives that appear to maintain and restore cellular homeostasis (Fig. 6). In the present study, we found that NO₂-CLA also regulated critical signaling pathways such as inflammation, angiogenesis, and antioxidant and heat shock responses that can be observed in the ECs treated with NO₂-OA or NO₂-LA. Noteworthy, we further identified novel genes and signaling pathways regulated by NO₂-CLA in ECs (Fig. 3 and Fig. 4, C and D), especially the pathways related to the response to hypoxia, fluid shear stress, and to atherosclerosis.

Fig. 6.

Summary of overrepresented pathways of DEGs regulated by NO₂-CLA in ECs. NO₂-CLA has unique effects on signaling pathways that are involved in EC homeostasis such as redox signaling, angiogenesis, response to hypoxia, and fluid shear stress. Furthermore, NO₂-CLA inhibits inflammatory gene expression in ECs under conditions of palmitic acid overload.

In ECs, NO₂-FAs enhance NO bioavailability (29, 31), inhibit inflammation (21, 57) and oxidative inflammatory reactions (19, 25, 27, 29, 67), enhance the heat shock response (25), and promote angiogenesis (45). NO₂-OA and NO₂-LA inhibit NF-κB activation and TNF-α-stimulated proinflammatory cytokine expression (10, 21). Fatty acid nitroalkenes also inhibit LPS-induced inflammation by suppressing the recruitment of TLR4 and TNF receptor-associated factor 6 (TRAF6) to EC lipid rafts (57). Elevated saturated palmitic acid levels are positively associated with metabolic disease and CVD (5, 52). Partial replacement of saturated FA-rich foods with those rich in cis-polyunsaturated FAs lowers the risk of hypertension and coronary heart disease (6, 40). Our transcriptome analysis further suggests that NO₂-CLA potently inhibits inflammation including the activation of NF-κB, TNF-α, and leukocyte-EC binding pathways that are activated by palmitic acid (Fig. 5, B and C).

Oxidative inflammatory reactions that are induced by dysregulated generation and scavenging of reactive species promote endothelial dysfunction and CVD (19). NO₂-LA and NO₂-OA dramatically induce the expression of HMOX1, a heme catabolism enzyme responsive to inflammatory stimuli and oxidative stress (29, 67). NO₂-OA upregulates HMOX1 expression in both NF-E2-related factor 2 (NRF2)-dependent and -independent mechanisms (25, 66). Also, NO₂-OA upregulates the antioxidant enzyme NAD(P)H quinone dehydrogenase 1 (NQO1) (25) and inhibits the activity of the pro-oxidant enzyme XOR in ECs (27). In the present study, we found that NO₂-CLA is not only a potent inducer of the antioxidant genes HMOX1 and NQO1 but also a suppressor of the pro-oxidant gene NADPH oxidase 4, emphasizing a critical role of NO₂-CLA in preserving EC redox homeostasis (Fig. 4C). Notably, NO₂-CLA also upregulates sequestosome 1/p62, which interacts with Kelch-like ECH-associated protein 1 and further activates NRF2 in response to oxidative stress (22) (Fig. 4C). Thus, these data significantly expand the understanding of how electrophilic NO₂-FA can modulate transcriptional responses of proteins critical to the preservation of vascular EC metabolic and adaptive signaling responses.

Dysregulated endothelin signaling is involved in the pathogenesis of various CVDs such as atherosclerosis, hypertension, and restenosis (50). NO₂-OA increases the NRF2-dependent expression of endothelin receptor type B in ECs and in turn reduces the extracellular concentration of EDN1 (26). We found that NO₂-CLA also significantly downregulates EDN1 gene expression (Fig. 3, A and B), suggesting that NO₂-FA can potentially beneficially impact endothelin signaling in various CVDs. NO₂-OA and NO₂-LA promote EC migration, sprouting, and angiogenesis (45). Among the genes validated in Fig. 3, MMP1 was significantly upregulated by NO₂-CLA in ECs. MMP1 is a protease that degrades collagen in the extracellular matrix and enhances vascular remodeling (62). In ECs, MMP1 upregulates VEGFR2 (38) and promotes angiogenesis (48). Our data suggest a potential mechanism that mediates the NO₂-FA-induced angiogenesis. OSGIN1 is an oxidative stress-induced gene that is upregulated by the transcription factor Nrf2 (34). Recent studies revealed that OSGIN1 enhances autophagy in the human airway epithelium (54, 61). Given NO₂-CLA-dependent upregulation of OSGIN1, there may be a potential role of NO₂-CLA in autophagy in ECs.

Cell cycle regulatory pathways such as cell cycle DNA replication, cell cycle checkpoint, and cell cycle phase transition are among the top enriched pathways in response to NO₂-CLA (Fig. 4, A and B). The accurate cell cycle transition from G1 phase to S phase is crucial for the control of cell proliferation (2). In smooth muscle cells, NO₂-LA inhibits cell proliferation via activation of NRF2 and upregulation of p27 (60). Moreover, in cultured pulmonary artery smooth muscle cells, NO₂-OA also suppresses cell proliferation (30). However, the role of NO₂-FAs in EC proliferation and the underlying mechanisms remain to be explored. The heat shock response is another major pathway activated by NO₂-OA, in which numerous heat shock genes were dramatically increased in ECs (25). Enriched pathway analysis revealed that NO₂-CLA consistently enhances the heat shock response (Fig. 4A), reinforcing the notion that heat shock response modulation contributes to the protective effect of NO₂-FAs in ECs.

Hypoxia leads to abnormal cell metabolism (13), impaired cell growth and survival, and angiogenesis (64). NO₂-OA and NO₂-LA promote EC migration, tube formation, and angiogenesis in a NO-hypoxia inducible factor-1α (HIF-1α)-dependent manner (45). The present data affirm that NO₂-CLA also regulates hypoxic responses via increased angiopoietin-like 4 (ANGPTL4) expression in ECs (Fig. 4C). ANGPTL4, in turn, promotes angiogenesis (32) and protects against myocardial infarction (18). CASPASE 1 (CASP1), a critical component of the inflammasome, was also identified as a novel gene downregulated by NO₂-CLA (Fig. 4C). Activated caspase-1 cleaves prointerleukin-1β (IL-1β) and pro-IL-18 into mature proinflammatory cytokines IL-1β and IL-18, respectively. The inhibition of caspase-1 increased postischemic angiogenesis in mice (36). Thus, the present data support a role for NO₂-CLA in modulating hypoxic responses associated with CVDs.

Additional novel pathways being regulated by NO₂-CLA were identified in the present study. Different shear stress patterns induce distinct phenotypes and functions associated with resistance/susceptibility to vascular pathology in the arterial ECs (11). Disturbed flow is proatherogenic by impairing redox homeostasis and promoting inflammation, whereas steady laminar blood flow is vasoprotective by regulating EC redox homeostasis and inflammation in the vascular wall (7). Indeed, different patterns of blood flow regulate distinct transcriptomes through mechanical sensors and the modulation of signaling pathways in ECs (41, 43). The current data reveal that NO₂-CLA regulates a cluster of genes that are responsive to fluid shear stress (Fig. 4, C and D). For example, laminar shear stress dramatically induces transcription factor KLF2, which upregulates the expression of anti-inflammatory, antioxidant, and antithrombotic genes that act to maintain vascular homeostasis (16). BMPs have been implicated in disorders ranging from hereditary hemorrhagic telangiectasia and peripheral arterial hypertension to atherosclerosis (14). Laminar flow inhibits BMP4, and in contrast, disturbed flow induces BMP4 expression in ECs (9, 53). Notably, similar to the effect of laminar shear stress, NO₂-CLA upregulates KLF2 and downregulates BMP4 in ECs. Also, these data suggest that NO₂-CLA upregulates GPC1 expression in ECs (Fig. 4, C and D). GPC1 acts as an FGF co-receptor to facilitate the binding of FGF to the FGF receptor (FGFR), subsequently activating FGF-FGFR signaling (44, 70). In ECs, GPC1 mediates the laminar flow-induced activation of the endothelial NO synthase (15, 68), supporting a potentially protective role of GPC1 in atherosclerosis. The mechanisms mediating the regulation of these novel targets by NO₂-CLA remain to be explored.

In summary, our study presents a comprehensive and systematic analysis of the endothelial cell transcriptome that responds uniquely to NO₂-CLA under basal and saturated FA-induced stress responses. NO₂-CLA beneficially regulates critical cellular pathways and responses including cell cycle, hypoxic, anti-inflammatory, and antioxidant responses. We also reveal novel NO₂-CLA-responsive genes and pathways, thereby expanding our understanding of potential NO₂-CLA effects in CVD, motivating new experimental pursuits and providing new insight into the underlying mechanisms that account for the effects of fatty acid nitroalkenes in the regulation of EC function.

GRANTS

This work was supported in part by National Institutes of Health Grants R01HL-138094 (to Y. Fan), R01HL-068878 (to Y. E. Chen), R01HL-123333 (to L. Villacorta), R01HL-64937, R01HL-132550, and R01HL-103455 (to B.A. Freeman) and GM-125944 and R01DK-112854 (to F.J. Schopfer), R01HL-138139 (to J. Zhang) and American Heart Association Grants 17GRN33660955 (to F.J. Schopfer), 17PRE33400179 (to H. Lu), and 18PRE34000005 (to W. Liang).

DISCLOSURES

B. A. Freeman, F. J. Schopfer, and Y. E. Chen acknowledge an interest in Complexa, Inc. The remaining authors declare no conflicts of interest, financial or otherwise.

AUTHOR CONTRIBUTIONS

H.L. and Y.F. conceived and designed research; H.L., J.S., and Y.F. performed experiments; H.L., J.S., W.L., J.Z., and Y.F. analyzed data; H.L., J.S., W.L., J.Z., S.L., L.V., F.J.S., B.A.F., Y.E.C., and Y.F. interpreted results of experiments; H.L. and J.S. prepared figures; H.L. and Y.F. drafted manuscript; H.L., J.S., W.L., J.Z., O.R., M.T.G.-B., S.L., L.V., F.J.S., B.A.F., Y.E.C., and Y.F. approved final version of manuscript; J.Z., O.R., M.T.G.-B., S.L., L.V., F.J.S., B.A.F., Y.E.C., and Y.F. edited and revised manuscript.